Unique associated Kaposi&#39;s sarcoma virus sequences and uses thereof

ABSTRACT

This invention provides an isolated DNA molecule which is at least 30 nucleotides in length and which uniquely defines a herpesvirus associated with Kaposi&#39;s sarcoma. This invention provides an isolated herpesvirus associated with Kaposi&#39;s sarcoma. This invention provides an antibody specific to the peptide. Antisense and triplex oligonucleotide molecules are also provided. This invention provides a method of vaccinating a subject for KS, prophylaxis diagnosing or treating a subject with KS and detecting expression of a DNA virus associated with Kaposi&#39;s sarcoma in a cell.

The invention disclosed herein was made with Government support under a co-operative agreement CCU210852 from the Centers for Disease Control and Prevention, of the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention.

This application is a 371 of PCT/US95/10194 filed Aug. 11, 1995, and continuation-in-part application of U.S. Ser. No. 08/420,235, filed on Apr. 11, 1995, U.S. Pat. No. 5,801,042, which is a continuation-in-part application of U.S. Ser. No. 08/343,101, filed on Nov. 21, 1994, U.S. Pat. No. 5,830,759, which is a continuation-in-part application of U.S. Ser. No. 08/292,365, filed on Aug. 18, 1994, abandoned, which is hereby incorporated by reference.

Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of each Experimental Details Section. The disclosures of the publications cited herein are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Kaposi's sarcoma (KS) is the most common neoplasm occurring in persons with acquired immunodeficiency syndrome (AIDS). Approximately 15-20% of AIDS patients develop this neoplasm which rarely occurs in immunocompetent individuals [13, 14]. Epidemiologic evidence suggests that AIDS-associated KS (AIDS-KS) has an infectious etiology. Gay and bisexual AIDS patients are approximately twenty times more likely than hemophiliac AIDS patients to develop KS, and KS may be associated with specific sexual practices among gay men with AIDS [6, 15, 55, 83]. KS is uncommon among adult AIDS patients infected through heterosexual or parenteral HIV transmission, or among pediatric AIDS patients infected through vertical HIV transmission [77]. Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, Epstein-Barr virus, human herpesvirus 6, human immunodeficiency virus (HIV), and Mycoplasma penetrans [18, 23, 85, 91, 92]. Non-infectious environmental agents, such as nitrite inhalants, also have been proposed to play a role in KS tumorigenesis [33]. Extensive investigations, however, have not demonstrated an etiologic association between any of these agents and AIDS-KS [37, 44, 46, 90].

SUMMARY OF THE INVENTION

This invention provides an isolated DNA molecule which is at least 30 nucleotides in length and which uniquely defines a herpesvirus associated with Kaposi's sarcoma. This invention provides an isolated herpesvirus associated with Kaposi's sarcoma.

This invention provides a method of vaccinating a subject for KS, prophylaxis diagnosing or treating a subject with KS and detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Agarose gel electrophoresis of RDA products from AIDS-KS tissue and uninvolved tissue. RDA was performed on DNA extracted from KS skin tissue and uninvolved normal skin tissue obtained at autopsy from a homosexual man with AIDS-KS. Lane 1 shows the initial PCR amplified genomic representation of the AIDS-KS DNA after Bam HI digestion. Lanes 2-4 show that subsequent cycles of ligation, amplification, hybridization and digestion of the RDA products resulted in amplification of discrete bands at 380, 450, 540 and 680 bp. RDA of the extracted AIDS-KS DNA performed against itself resulted in a single band at 540 bp (lane 5). Bands at 380 bp and 680 bp correspond to KS330Bam and KS627Bam respectively after removal of 28 bp priming sequences. Bands at 450 and 540 bp hybridized nonspecifically to both KS and non-KS human DNA. Lane M is a molecular weight marker.

FIGS. 2A-2B: Hybridization of ³² P-labelled KS330Bam (FIG. 2A) and KS627Bam (FIG. 2B) sequences to a representative panel of 19 DNA samples extracted from KS lesions and digested with Bam HI.

KS330Bam hybridized to 11 of the 19 and KS627Bam hybridized to 12 of the 19 DNA samples from AIDS-KS lesions. Two additional cases (lanes 12 eand 13) were shown to have faint bands with both KS330Bam and KS627Bam probes after longer exposure. One negative specimen (lane 3) did not have microscopically detectable KS in the tissue specimen. Seven of 8 additional KS DIA samples also hybridized to both sequences.

FIG. 3A-3F: Nucleotide sequences of the DNA herpesvirus associated with KS (KSHV).

FIG. 4A-4B: PCR amplification of a representative set of KS-derived DNA samples using KS330₂₃₄ primers. FIG. 4A shows the agarose gel of the amplification products from 19 KS DNA samples (lanes 1-19) and FIG. 4B shows specific hybridization of the PCR products to a ³² P end-labelled 25 bp internal oligonucleotide (FIG. 3B) after transfer of the gel to a nitrocellulose filter. Negative samples in lanes 3 and 15 respectively lacked microscopically detectable KS in the sample or did not amplify the constitutive p53 exon 6, suggesting that these samples were negative for technical reasons. An additional 8 AIDS-KS samples were amplified and all were positive for KS330₂₃₄. Lane 20 is a negative control and Lane M is a molecular weight marker.

FIG. 5: Southern blot hybridization of KS330Bam and KS627Bam to AIDS-KS genomic DNA extracted from three subjects (lanes 1, 2, and 3) and digested with PvuII. Based on sequence information (FIG. 3A), restricted sites for Pvu II occur between bp 12361-12362 of the KSHV sequence (FIG. 3A, SEQ ID NO: 1), at bp 134 in KS330Bam (FIG. 3B, SEQ ID NO: 2) and bp 414 in KS627Bam (FIG. 3C, SEQ ID NO: 3). KS330Bam and KS627Bam failed to hybridize to the same fragments in the digests indicating that the two sequences are separated from each other by one or more intervening Bam HI restriction fragments. Digestion with Pvu II and hybridization to KS330Bam resulted in two distinct banding patterns (lanes 1 and 2 vs. lane 3) suggesting variation between KS samples.

FIG. 6: Comparison of amino acid homologies between EBV ORF BDLF1 (SEQ ID NO: 47), and HSVA ORF 26 (SEQ ID NO: 46) and a 918 bp reading frame of the Kaposi's sarcoma agent which includes KS330Bam. Amino acid identity is denoted by reverse lettering. In HSVSA, ORF 26 encodes a minor capsid VP23 which is a late gene product.

FIG. 7: Subculture of Raji cells co-cultivated with BCBL-1 cells treated with TPA for 2 days. PCR shows that Raji cells are positive for KSHV sequences and indicate that the agent is a transmissible virus.

FIG. 8: A schematic diagram of the orientation of KSHV open reading frames identified on the KS5 20,710 bp DNA fragment. Homologs to each open reading frame from a corresponding region of the herpesvirus saimiri (HSVSA) genome are present in an identical orientation, except for the region corresponding to the ORF 28 of HSVSA (middle schematic section). The shading for each open reading frame corresponds to the approximate % amino acid identity for the KSHV ORF compared to this homolog in HSVSA. Noteworthy homologs that are present in this section of DNA include homologs to thymidine kinase (ORF21), gH glycoprotein (ORF22), major capsid protein (ORF25) and the VP23 protein (ORF26) which contains the original KS330Bam sequence derived by representational difference analysis.

FIG. 9: The ˜200 kD antigen band appearing on a Western blot of KS patient sera against BCBL1 lysate (B1) and Raji lysate (RA). M is molecular weight marker. The antigen is a doublet between ca. 210 kD and 240 kD.

FIG. 10: 5 control patient sera without KS (A1N, A2N, A3N, A4N and A5N). B1=BCBL1 lysate, RA=Raji lysate. The 220 kD band is absent from the Western blots using patient sera without KS.

FIG. 11: In this figure, 0.5 ml aliquots of the gradient have been fractionated (fractions 1-62) with the 30% gradient fraction being at fraction No. 1 and the 10% gradient fraction being at fraction No. 62. Each fraction has been dot hybridized to a nitrocellulose membrane and then a ³² P-labeled KSHV DNA fragment, KS631Bam has been hybridized to the membrane using standard techniques. The figure shows that the major solubilized fraction of the KSHV genome bands (i.e. is isolated) in fractions 42 through 48 of the gradient with a high concentration of the genome being present in fraction 44. A second band of solubilized KSHV DNA occurs in fractions 26 through 32.

FIG. 12: Location, feature, and relative homologies of KS5 open reading frames compared to translation products of herpesvirus saimiri (HSV), equine herpesvirus 2 (EHV2) and Epstein-Barr virus (EBV).

FIG. 13: Indirect immunofluorescence end-point and geometric mean titers (GMT) in AIDS-KS and AIDS control sera against HBL-6 and P3H3 prior to and after adsorption with P3H3.

FIG. 14: Genetic map of KS5, a 20.7 kb lambda phage clone insert derived from a human genomic library prepared from an AIDS-KS lesion. Seventeen partial and complete open reading frames (ORFs) are identified with arrows denoting reading frame orientations. Comparable regions of the Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) genomes are shown for comparison. Levels of amino acid similarity between KSHV ORFs are indicated by shading of EBV and HVS ORFs (black, over 70% similarity; dark gray, 55-70% similarity; light gray, 40-54% similarity; white, no detectable homology). Domains of conserved herpesvirus sequence blocks and locations of restriction endonuclease sites used in subcloning are shown beneath the KSHV map (B, Bam HI site; N, Not I site). The small Bam HI fragment (black) in the VP23 gene homolog corresponds to the KS330Bam fragment generated by representational difference analysis which was used to identify the KS5 lambda phage clone.

FIGS. 15A-15B: Phylogenetic trees of KSHV based on comparison of aligned amino acid sequences between herpesviruses for the MCP gene and for a concatenated nine-gene set. The comparison of MCP sequences (FIG. 15A) was obtained by the neighbor-joining method and is shown in unrooted form with branch lengths proportional to divergence (mean number of substitution events per site) between the nodes bounding each branch. Comparable results were obtained by maximum parsimony analysis. The number of times out of 100 bootstrap samplings the division indicated by each internal branch was obtained are shown next to each branch; bootstrap values below 75 are not shown. FIG. 15B is a phylogenetic tree of gammaherpesvirus sequences based on a nine-gene set CS1 (see text) and demonstrates that KSHV is most closely related to the gamma-2 herpesvirus sublineage, genus Rhadinovirus. The CS1 amino acid sequence was used to infer a tree by the Protml maximum likelihood method; comparable results, not shown were obtained with the neighbor-joining and maximum parsimony methods. The bootstrap value for the central branch is marked. On the basis of the MCP analysis, the root must lie between EBV and the other three species. Abbreviations for virus species used in the sequence comparisons are 1) Alphaherpesvirinae: HSV1 and HSV2, herpes simplex virus types 1 and 2; EHV1, equine herpesvirus 1; PRV, pseudorabies virus; and VZV, varicella-zoster virus, 2) Betaherpesvirinae: HCMV, human cytomegalovirus; HHV6 and HHV7, human herpesviruses 6 and 7, and 3) Gammaherpesvirinae: HVS, herpesvirus saimiri; EHV2, equine herpesvirus 2; EBV, Epstein-Barr virus; and Kaposi's sarcoma-associated herpesvirus.

FIGS. 16A-16B: CHEF gel electrophoresis of BCBL-1 DNA hybridized to KS631Bam (FIG. 16A) and EBV terminal repeat (FIG. 16B). KS631Bam hybridizes to a band at 270 kb as well as to a diffuse band at the origin. The EBV termini sequence hybridizes to a 150-160 kb band consistent with the linear form of the genome. Both KS631Bam (dark arrow) and an EBV terminal sequence hybridize to high molecular weight bands immediately below the origin indicating possible concatemeric or circular DNA. The high molecular weight KS631Bam hybridizing band reproduces poorly but is visible on the original autoradiographs.

FIG. 17: Induction of KSHV and EBV replication in BCBL-1 with increasing concentrations of TPA. Each determination was made in triplicate after 48 h of TPA incubation and hybridization was standardized to the amount of cellular DNA by hybridization to beta-actin. The figure shows the mean and range of relative increase in hybridizing genome for EBV and KSHV induced by TPA compared to uninduced BCBL-1. TPA at 20 ng/ml induced an eight-fold increase in EBV genome (upper line) at 48 h compared to only a 1.4 fold increase in KSHV genome (lower line).

Despite the lower level of KSHV induction, increased replication of KSHV genome after induction with TPA concentrations over 10 ng/ml was reproducibly detected.

FIGS. 18A-18C: In situ hybridization with an ORF26 oligomer to BCBL-1, Raji and RCC-1 cells. Hybridization occurred to nuclei of KSHV infected BCBL-1 (FIG. 18A), but not to uninfected Raji cells (FIG. 18B). RCC-1, a Raji cell line derived by cultivation of Raji with BCBL-1 in communicating chambers separated by a 0.45μ filter, shows rare cells with positive hybridization to the KSHV ORF26 probe (FIG. 18C).

FIGS. 19A-19D: Representative example of IFA staining of HBL-6 with AIDS-KS patient sera and control sera from HIV-infected patients without KS. Both AIDS-KS (FIG. 19A) and control (FIG. 19B) sera show homogeneous staining of HBL-6 at 1:50 dilution. After adsorption with paraformaldehyde-fixed P3H3 to remove cross-reacting antibodies directed against lymphocyte and EBV antigens, antibodies from AIDS-KS sera localize to HBL-6 nuclei (FIG. 19C). P3H3 adsorption of control sera eliminates immunofluorescent staining of HBL-6 (FIG. 19D).

FIGS. 20A-20B: Longitudinal PCR examination for KSHV DNA of paired PBMC samples from AIDS-KS patients (A) and homosexual/bisexual AIDS patients without KS (B). Time 0 is the date of KS onset for cases; or other AIDS-defining illness for controls. All samples were randomized and examined blindly. Overall, 7 of the KS patients were KSHV positive at both examination dates (solid bars) and 5 converted from a negative to positive PBMC sample (forward striped bars) immediately prior to or after KS onset. Two previously positive KS patients were negative after KS diagnosis (reverse striped bars) and the remaining KS patients were negative at both timepoints (open bars). Two homosexual/bisexual control PBMC samples without KS converted from negative to positive and one control patient reverted from PCR positive to negative for KSHV DNA.

FIG. 21: Sample collection characteristics for AIDS-KS patients, gay/bisexual AIDS patients and hemophilic AIDS patients.

FIG. 22: PCR analysis of KS330₂₃₃ in DNA samples from patients with Kaposi's sarcoma and tumor controls.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C=cytosine A=adenosine

T=thymidine G=guanosine

The term "nucleic acids", as used herein, refers to either DNA or RNA. "Nucleic acid sequence" or "polynucleotide sequence" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and nonfunctional DNA or RNA.

By a nucleic acid sequence "homologous to" or "complementary to", it is meant a nucleic acid that selectively hybridizes, duplexes or binds to viral DNA sequences encoding proteins or portions thereof when the DNA sequences encoding the viral protein are present in a human genomic or cDNA library. A DNA sequence which is homologous to a target sequence can include sequences which are shorter or longer than the target sequence so long as they meet the functional test set forth. Hybridization conditions are specified along with the source of the CDNA library.

Typically, the hybridization is done in a Southern blot protocol using a 0.2XSSC, 0.1% SDS, 65° C. wash. The term "SSC" refers to a citrate-saline solution of 0.15 M sodium chloride and 20 Mm sodium citrate. Solutions are often expressed as multiples or fractions of this concentration. For example, 6×SSC refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate. 0.2×SSC refers to a solution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4 mM sodium citrate.

The phrase "nucleic acid molecule encoding" refers to a nucleic acid molecule which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid molecule include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

The phrase "expression cassette", refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible with such sequences. Such cassettes include at least promoters and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.

The term "operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.

The term "vector", refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector," this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types. Where a recombinant microorganism or cell culture is described as hosting an "expression plasmid", this includes latent viral DNA integrated into the host chromosome(s). Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cells during mitosis as an autonomous structure or is incorporated within the host's genome.

The phrase "recombinant protein" or "recombinantly produced protein" refers to a peptide or protein produced using non-native cells that do not have an endogenous copy of DNA able to express the protein. The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein.

The following terms are used to describe the sequence relationships between two or more nucleic acid molecules or polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence.

Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

As applied to polypeptides, the terms "substantial identity" or "substantial sequence identity" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap which share at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 99 percent sequence identity or more.

"Percentage amino acid identity" or "percentage amino acid sequence identity" refers to a comparison of the amino acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same amino acids. For example, "95% amino acid identity" refers to a comparison of the amino acids of two polypeptides which when optimally aligned have 95% amino acid identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.

The phrase "substantially purified" or "isolated" when referring to a herpesvirus peptide or protein, means a chemical composition which is essentially free of other cellular components. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80% of all macromolecular species present in the preparation. Preferably, the protein is purified to represent greater than 90% of all macromolecular species present. More preferably the protein is purified to greater than 95%, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.

The phrase "specifically binds to an antibody" or "specifically immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the herpesvirus of the invention in the presence of a heterogeneous population of proteins and other biologics including viruses other than the herpesvirus. Thus, under designated immunoassay conditions, the specified antibodies bind to the herpesvirus antigens and do not bind in a significant amount to other antigens present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the human herpesvirus immunogen described herein can be selected to obtain antibodies specifically immunoreactive with the herpesvirus proteins and not with other proteins. These antibodies recognize proteins homologous to the human herpesvirus protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane [32] for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

"Biological sample" as used herein refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids and tissue specimens.

I. Kaposis's Sarcoma (KS)--Associated Herpesvirus.

This invention provides an isolated DNA molecule which is at least 30 nucleotides in length and which uniquely defines a herpesvirus associated with Kaposi's sarcoma.

In one embodiment the isolated DNA molecule comprises at least a portion of the nucleic acid sequence as shown in FIG. 3A (SEQ ID NO: 1). In another embodiment the isolated DNA molecule is a 330 base pair (bp) sequence. In another embodiment the isolated DNA molecule is a 12-50 bp sequence. In another embodiment the isolated DNA molecule is a 30-37 bp sequence.

In another embodiment the isolated DNA molecule is genomic DNA. In another embodiment the isolated DNA molecule is cDNA. In another embodiment a RNA is derived form the isolated nucleic acid molecule or is capable of hybridizing with the isolated DNA molecule. As used herein "genomic" means both coding and non-coding regions of the isolated nucleic acid molecule.

Further, the DNA molecule above may be associated with lymphoproliferative diseases including, but not limited to: Hodgkin's disease, non-Hodgkin's lymphoma, lymphatic leukemia, lymphosarcoma, splenomegaly, reticular cell sarcoma, Sezary's syndrome, mycosis fungoides, central nervous system lymphoma, AIDS related central nervous system lymphoma, post-transplant lymphoproliferative disorders, and Burkitt's lymphoma. A lymphoproliferative disorder is characterized as being the uncontrolled clonal or polyclonal expansion of lymphocytes involving lymph nodes, lymphoid tissue and other organs.

This invention provides an isolated nucleic acid molecule encoding an ORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ ID NOs: 14 and 15), ORF22 (SEQ ID NOs: 16 and 17), ORF23 (SEQ ID NOs: 18 and 19), ORF24 (SEQ ID NOs: 20 and 21), ORF25 (SEQ ID NOs: 2 and 3), ORF26 (SEQ ID NOs: 24 and 25), ORF27 (SEQ ID NOs: 26 and 27), ORF28 (SEQ ID NOs: 28 and 29), ORF29A (SEQ ID NOs: 30 and 31), ORF29B (SEQ ID NOs: 4 and 5), ORF30 (SEQ ID NOs: 6 and 7), ORF31 (SEQ ID NOs: 8 and 9), ORF32 (SEQ ID NOs: 32 and 33), ORF33 (SEQ ID NOs: 10 and 11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ ID NOs: 12 AND 13).

This invention provides an isolated polypeptide encoded by ORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ ID NOs: 14 and 15), ORF22 (SEQ ID NOs: 16 and 17), ORF23 (SEQ ID NOs: 18 and 19), ORF24 (SEQ ID NOs: 20 and 21), ORF25 (SEQ ID NOs: 2 and 3), ORF26 (SEQ ID NOs: 24 and 25), ORF27 (SEQ ID NOs: 26 and 27), ORF28 (SEQ ID NOs: 28 and 29), ORF29A (SEQ ID NOs: 30 and 31), ORF29B (SEQ ID NOs: 4 and 5), ORF30 (SEQ ID NOs: 6 and 7), ORF31 (SEQ ID NOs: 8 and 9), ORF32 (SEQ ID NOs: 32 and 33), ORF33 (SEQ ID NOs: 10 and 11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ ID NOs: 12 AND 13).

For Example, TK is encoded by ORF 21; glycoprotein H (gH) by ORF 22; major capsid protein (MCP) by ORF 25; virion polypeptide (VP23) by ORF 26; and minor capsid protein by ORF 27.

This invention provides for a replicable vector comprising the isolated DNA molecule of the DNA virus. The vector includes, but is not limited to: a plasmid, cosmid, λ phage or yeast artificial chromosome (YAC) which contains at least a portion of the isolated nucleic acid molecule.

As an example to obtain these vectors, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with DNA ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available and known to an ordinary skilled practitioner.

Regulatory elements required for expression include promoter or enhancer sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art, for example the methods described above for constructing vectors in general.

This invention provides a host cell containing the above vector. The host cell may contain the isolated DNA molecule artificially introduced into the host cell. The host cell may be a eukaryotic or bacterial cell (such as E.coli), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

This invention provides an isolated herpesvirus associated with Kaposi's sarcoma. In one embodiment the herpesvirus comprises at least a portion of a nucleotide sequence as shown in FIGS. 3A (SEQ ID NO: 1).

In one embodiment the herpesvirus may be a DNA virus. In another embodiment the herpesvirus may be a Herpesviridae. In another embodiment the herpesvirus may be a gammaherpesvirinae. The classification of the herpesvirus may vary based on the phenotypic or molecular characteristics which are known to those skilled in the art.

This invention provides an isolated DNA virus wherein the viral DNA is about 270 kb in size, wherein the viral DNA encodes a thymidine kinase, and wherein the viral DNA is capable of selectively hybridizing to a nucleic acid probe selected from the group consisting of SEQ ID NOs: 38-40.

The KS-associated human herpesvirus of the invention is associated with KS and is involved in the etiology of the disease. The taxonomic classification of the virus has not yet been made and will be based on phenotypic or molecular characteristics known to those of skill in the art. However, the novel KS-associated virus is a DNA virus that appears to be related to the Herpesviridae family and the gammaherpesvirinae subfamily, on the basis of nucleic acid homology.

A. Sequence identity of the viral DNA and its proteins.

The human herpesvirus of the invention is not limited to the virus having the specific DNA sequences described herein. The KS-associated human herpesvirus DNA shows substantial sequence identity, as defined above, to the viral DNA sequences described herein. DNA from the human herpesvirus typically selectively hybridizes to one or more of the following three nucleic acid probes:

    Probe 1 (SEQ ID NO:38)                                                          AGCCGAAAGG ATTCCACCAT TGTGCTCGAA TCCAACGGAT                                   TTGACCCCGT GTTCCCCATG GTCGTGCCGC AGCAACTGGG                                      - GCACGCTATT CTGCAGCAGC TGTTGGTGTA CCACATCTAC                                  - TCCAAAATAT CGGCCGGGGC CCCGGATGAT GTAAATATGG                                  - CGGAACTTGA TCTATATACC ACCAATGTGT CATTTATGGG                                  - GCGCACATAT CGTCTGGACG TAGACAACAC GGA                                         - Probe 2 (SEQ ID NO:39):                                                     GAAATTACCC ACGAGATCGC TTCCCTGCAC ACCGCACTTG                                     - GCTACTCATC AGTCATCGCC CCGGCCCACG TGGCCGCCAT                                  - AACTACAGAC ATGGGAGTAC ATTGTCAGGA CCTCTTTATG                                  - ATTTTCCCAG GGGACGCGTA TCAGGACCGC CAGCTGCATG                                  - ACTATATCAA AATGAAAGCG GGCGTGCAAA CCGGCTCACC                                  - GGGAAACAGA ATGGATCACG TGGGATACAC TGCTGGGGTT                                  - CCTCGCTGCG AGAACCTGCC CGGTTTGAGT CATGGTCAGC                                  - TGGCAACCTG CGAGATAATT CCCACGCCGG TCACATCTGA                                  - CGTTGCCT                                                                     - Probe 3 (SEQ ID NO:40):                                                     AACACGTCAT GTGCAGGAGT GACATTGTGC CGCGGAGAAA                                     - CTCAGACCGC ATCCCGTAAC CACACTGAGT GGGAAAATCT                                  - GCTGGCTATG TTTTCTGTGA TTATCTATGC CTTAGATCAC                                  - AACTGTCACC CG                                                         

Hybridization of a viral DNA to the nucleic acid probes listed above is determined by using standard nucleic acid hybridization techniques as described herein. In particular, PCR amplification of a viral genome can be carried out using the following three sets of PCR primers:

    1)     AGCCGAAAGGATTCCACCAT;                                                                              (SEQ ID NO:41)                                           TCCGTGTTGTCTACGTCCAG (SEQ ID NO:48)                                          - 2) GAAATTACCCACGAGATCGC; (SEQ ID NO:42)                                        AGGCAACGTCAGATGTGA (SEQ ID NO:43)                                            - 3) AACACGTCATGTGCAGGAGTGAC; (SEQ ID NO:43)                                     CGGGTGACAGTTGTGATCTAAGG (SEQ ID NO:50)                               

In PCR techniques, oligonucleotide primers, as listed above, complementary to the two 3' borders of the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols: A Guide to Methods and Applications [74]. Following PCR amplification, the PCR-amplified regions of a viral DNA can be tested for their ability to hybridize to the three specific nucleic acid probes listed above. Alternatively, hybridization of a viral DNA to the above nucleic acid probes can be performed by a Southern blot procedure without viral DNA amplification and under stringent hybridization conditions as described herein.

Oligonucleotides for use as probes or PCR primers are chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Carruthers [19] using an automated synthesizer, as described in Needham-VanDevanter [69]. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J. D. and Regnier, F. E. [75A]. The sequence of the synthetic oligonucleotide can be verified using the chemical degradation method of Maxam, A. M. and Gilbert, W. [63].

B. Isolation and propagation of KS-inducing strains of the Human Herpesvirus

Using conventional methods, the human herpesvirus can be propagated in vitro. For example, standard techniques for growing herpes viruses are described in Ablashi, D. V. [1]. Briefly, PHA stimulated cord blood mononuclear cells, macrophage, neuronal, or glial cell lines are cocultivated with cerebrospinal fluid, plasma, peripheral blood leukocytes, or tissue extracts containing viral infected cells or purified virus. The recipient cells are treated with 5 μg/ml polybrene for 2 hours at 37° C. prior to infection.

Infected cells are observed by demonstrating morphological changes, as well as being positive for antigens from the human herpesvirus by using monoclonal antibodies immunoreactive with the human herpes virus in an immunofluorescence assay.

For virus isolation, the virus is either harvested directly from the culture fluid by direct centrifugation, or the infected cells are harvested, homogenized or lysed and the virus is separated from cellular debris and purified by standard methods of isopycnic sucrose density gradient centrifugation.

One skilled in the art may isolate and propagate the DNA herpesvirus associated with Kaposi's sarcoma (KSHV) employing the following protocol. Long-term establishment of a B lymphoid cell line infected with the KSHV from body-cavity based lymphomas (RCC-1 or HBL-6) is prepared extracting DNA from the Lymphoma tissue using standard techniques [27, 49, 66].

The KS associated herpesvirus may be isolated from the cell DNA in the following manner. An infected cell line (HBL-6 RCC-1), which can be lysed using standard methods such as hyposomatic shocking and Dounce homogenization, is first pelleted at 2000×g for 10 minutes, the supernatant is removed and centrifuged again at 10,000×g for 15 minutes to remove nuclei and organelles. The supernatant is filtered through a 0.45μ filter and centrifuged again at 100,000×g for 1 hour to pellet the virus. The virus car. then be washed and centrifuged again at 100,000 ×g for 1 hour.

The DNA is tested for the presence of the KSHV by Southern blotting and PCR using the specific probes as described hereinafter. Fresh lymphoma tissue containing viable infected cells is simultaneously filtered to form a single cell suspension by standard techniques [49, 66]. The cells are separated by standard Ficoll-Plaque centrifugation and lymphocyte layer is removed. The lymphocytes are then placed at >1×10⁶ cells/ml into standard lymphocyte tissue culture medium, such as RMP 1640 supplemented with 10% fetal calf serum. Immortalized lymphocytes containing the KSHV virus are indefinitely grown in the culture media while nonimmortilized cells die during course of prolonged cultivation.

Further, the virus may be propagated in a new cell line by removing media supernatant containing the virus from a continuously infected cell Line at a concentration of >1×10⁶ cells/ml. The media is centrifuged at 2000×g for 10 minutes and filtered through a 0.45μ filter to remove cells. The media is applied in a 1:1 volume with cells growing at >1×10⁶ cells/ml for 48 hours. The cells are washed and pelleted and placed in fresh culture medium, and tested after 14 days of growth.

RCC-1 and RCC-1₂ F5 were deposited on Oct. 19, 1994 under ATCC Accession No. CRL 11734 and CRL 11735, respectively, pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.

HBL-6 was deposited on Nov. 18, 1994 under ATCC Accession No. CRL 11762 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.

C. Immunological Identity of the Virus

The KS-associated human herpesvirus can also be described immunologically. KS-associated human herpesviruses are selectively immunoreactive to antisera generated against a defined immunogen such as the viral major capsid protein depicted in Seq. ID No. 12, herein. Immunoreactivity is determined in an immunoassay using a polyclonal antiserum which was raised to the protein which is encoded by the amino acid sequence or nucleic acid sequence of SEQ ID NOs: 18-20. This antiserum is selected to have low crossreactivity against other herpes viruses and any such crossreactivity is removed by immunoabsorbtion prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 18-20 is isolated as described herein. For example, recombinant protein can be produced in a mammalian cell line. An inbred strain of mice such as balb/c is immunized with the protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 2-37 using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see [32], supra). Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10⁴ or greater are selected and tested for their cross reactivity against other viruses of the gammaherpesvirinae subfamily, particularly human herpes virus types 1-7, by using a standard immunoassay as described in [32], supra. These other gammaherpesvirinae virus can be isolated by standard techniques for isolation herpes viruses as described herein.

The ability of the above viruses to compete with the binding of the antisera to the immunogen protein is determined. The percent crossreactivity for other viruses is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the other viruses listed above is selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed viruses.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay procedure as described above to compare an unknown virus preparation to the specific KS herpesvirus preparation described herein and containing the nucleic acid sequence described in SEQ ID NOs: 2-37. In order to make this comparison, the immunogen protein which is encoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 2-37 is the labeled antigen and the virus preparations are each assayed at a wide range of concentrations. The amount of each virus preparation required to inhibit 50% of the binding of the antisera to the labeled immunogen protein is determined. Those viruses that specifically bind to an antibody generated to an immunogen consisting of the protein of SEQ ID NOs: 2-37 are those virus where the amount of virus needed to inhibit 50% of the binding to the protein does not exceed an established amount. This amount is no more than 10 times the amount of the virus that is needed for 50% inhibition for the KS-associated herpesvirus containing the DNA sequence of SEQ ID NO: 1. Thus, the KS-associated herpesviruses of the invention can be defined by immunological comparison to the specific strain of the KS-associated herpesvirus for which nucleic acid sequences are provided herein.

This invention provides, a nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with the isolated DNA molecule. In one embodiment, the molecule is DNA. In another embodiment, the molecule is RNA. In another embodiment the nucleic acid molecule may be 14-20 nucleotides in length. In another embodiment the nucleic acid molecule may be 16 nucleotides in length.

This invention provides, a nucleic acid molecule of at least 14 nucleotides capable of specifically hybridizing with a nucleic acid molecule which is complementary to the isolated DNA molecule In one embodiment, the molecule is DNA. In another embodiment, the molecule is RNA.

The nucleic acid molecule of at least 14 nucleotides may hybridize with moderate stringency to at least a portion of a nucleic acid molecule with a sequence shown in FIGS. 3A-3F (SEQ ID NOs: 1, 10-17, and 38-40).

High stringent hybridization conditions are selected at about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60° C. As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents, ie. salt or formamide concentration, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one. For Example high stringency may be attained for example by overnight hybridization at about 68° C. in a 6× SSC solution, washing at room temperature with 6× SSC solution, followed by washing at about 680° in a 6× SSC in a 0.6× SSX solution.

Hybridization with moderate stringency may be attained for example by: 1) filter pre-hybridizing and hybridizing with a solution of 3× sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris buffer at Ph 7.5, 5× Denhardt's solution; 2.) pre-hybridization at 37° C. for 4 hours; 3) hybridization at 37° C. with amount of labelled probe equal to 3,000,000 cpm total for 16 hours; 4) wash in 2× SSC and 0.1% SDS solution; 5) wash 4× for 1 minute each at room temperature at 4× at 60° C. for 30 minutes each; and 6) dry and expose to film.

The phrase "selectively hybridizing to" refers to a nucleic acid probe that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a probe binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization. in a different "Complementary" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing Conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., [81] or Ausubel, F., et al., [8].

It will be readily understood by those skilled in the art and it is intended here, that when reference is made to particular sequence listings, such reference includes sequences which substantially correspond to its complementary sequence and those described including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the pathogenic organism or disease marker to which the relevant sequence listing relates.

Nucleic acid probe technology is well known to those skilled in the art who readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. DNA probe molecules may be produced by insertion of a DNA molecule having the full-length or a fragment of the isolated nucleic acid molecule of the DNA virus into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.

DNA virus nucleic acid rearrangements/mutations may be detected by Southern blotting, single stranded conformational polymorphism gel electrophoresis (SSCP), PCR or other DNA based techniques, or for RNA species by Northern blotting, PCR or other RNA-based techniques.

RNA probes may be generated by inserting the full length or a fragment of the isolated nucleic acid molecule of the DNA virus downstream of a bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with a linearized isolated nucleic acid molecule of the DNA virus or its fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.

As defined herein nucleic acid probes may be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, [19], or by the triester method according to Matteucci, et al., [62], both incorporated herein by reference. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid. It is also understood that when a specific sequence is identified for use a nucleic probe, a subsequence of the listed sequence which is 25 basepairs or more in length is also encompassed for use as a probe.

The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms. These molecules include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.

This invention provides for an isolated DNA molecule which encodes at least a portion of a Kaposi's sarcoma associated herpesvirus: virion polypeptide 23, major capsid protein, capsid proteins, thymidine kinase, or tegument protein.

This invention also provides a method of producing a polypeptide encoded by isolated DNA molecule, which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention provides an isolated peptide encoded by the isolated DNA molecule associated with Kaposi's sarcoma. In one embodiment the peptide may be a polypeptide. Further, this invention provides a host cell which expresses the polypeptide of isolated DNA molecule.

In one embodiment the isolated peptide or polypeptide is encoded by at least a portion of an isolated DNA molecule. In another embodiment the isolated peptide or polypeptide is encoded by at least a portion of a nucleic acid molecule with a sequence as set forth in (SEQ ID NOs: 2-37).

Further, the isolated peptide or polypeptide encoded by the isolated DNA molecule may be linked to a second nucleic acid molecule to form a fusion protein by expression in a suitable host cell. In one embodiment the second nucleic acid molecule encodes beta-galactosidase. Other nucleic acid molecules which are used to form a fusion protein are known to those skilled in the art.

This invention provides an antibody which specifically binds to the peptide or polypeptide encoded by the isolated DNA molecule. In one embodiment the antibody is a monoclonal antibody. In another embodiment the antibody is a polyclonal antibody.

The antibody or DNA molecule may be labelled with a detectable marker including, but not limited to: a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker, or gold. Radioactive labels include, but are not limited to: ³ H, ¹⁴ C, ³² P, ³³ P; ³⁵ S, ³⁶ Cl, ⁵¹ Or, ⁵⁷ Co, ⁵⁹ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re. Fluorescent markers include but are not limited to: fluorescein, rhodamine and auramine. Colorimetric markers include, but are not limited to: biotin, and digoxigenin. Methods of producing the polyclonal or monoclonal antibody are known to those of ordinary skill in the art.

Further, the antibody or nucleic acid molecule complex may be detected by a second antibody which may be linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. Other enzymes which may be employed are well known to one of ordinary skill in the art.

This invention provides a method to select specific regions on the polypeptide encoded by the isolated DNA molecule of the DNA virus to generate antibodies. The protein sequence may be determined from the cDNA sequence. Amino acid sequences may be analyzed by methods well known to those skilled in the art to determine whether they produce hydrophobic or hydrophilic regions in the proteins which they build. In the case of cell membrane proteins, hydrophobic regions are well known to form the part of the protein that is inserted into the lipid bilayer of the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Usually, the hydrophilic regions will be more immunogenic than the hydrophobic regions. Therefore the hydrophilic amino acid sequences may be selected and used to generate antibodies specific to polypeptide encoded by the isolated nucleic acid molecule encoding the DNA virus. The selected peptides may be prepared using commercially available machines. As an alternative, DNA, such as a cDNA or a fragment thereof, may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced by immunizing animals using the selected peptides. Monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody. Alternatively, monoclonal antibodies may be produced by in vitro techniques known to a person of ordinary skill in the art. These antibodies are useful to detect the expression of polypeptide encoded by the isolated DNA molecule of the DNA virus in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.

II. Immunoassays

The antibodies raised against the viral strain or peptides may be detectably labelled, utilizing conventional labelling techniques well-known to the art. Thus, the antibodies may be radiolabelled using, for example, radioactive isotopes such as ³ H, ¹²⁵ I, ¹³¹ I, and ³⁵ S.

The antibodies may also be labelled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techniques known in the art. Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin, and Texas Red.

Since specific enzymes may be coupled to other molecules by covalent links, the possibility also exists that they might be used as labels for the production of tracer materials. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase. Two principal types of enzyme immunoassay are the enzyme-linked immunosorbent assay (ELISA), and the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT, Syva Corporation, Palo Alto, Calif.). In the ELISA system, separation may be achieved, for example, by the use of antibodies coupled to a solid phase. The EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.

Additionally, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, bioluminescent compounds may be utilized for labelling, the bioluminescent compounds including luciferin, luciferase, and aequorin.

Once labeled, the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide) utilizing techniques well-known to the art.

A description of a radioimmunoassay (RIA) may be found in Laboratory Techniques in Biochemistry and Molecular Biology [52], with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein.

A description of general immunometric assays of various types can be found in the following U.S. Pat. Nos. 4,376,110 (David et al.) or 4,098,876 Piasio).

A. Assays for viral antigens

In addition to the detection of the causal agent using nucleic acid hybridization technology, one can use immunoassays to detect for the virus, specific peptides, or for antibodies to the virus or peptides. A general overview of the applicable technology is in Harlow and Lane [32], incorporated by reference herein.

In one embodiment, antibodies to the human herpesvirus can be used to detect the agent in the sample. In brief, to produce antibodies to the agent or peptides, the sequence being targeted is expressed in transfected cells, preferably bacterial cells, and purified. The product is injected into a mammal capable of producing antibodies. Either monoclonal or polyclonal antibodies (as well as any recombinant antibodies) specific for the gene product can be used in various immunoassays. Such assays include competitive immunoassays, radioimmunoassays, Western blots, ELISA, indirect immunofluorescent assays and the like. For competitive immunoassays, see Harlow and Lane [32] at pages 567-573 and 584-589.

Monoclonal antibodies or recombinant antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells or other lymphocytes from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein [50], incorporated herein by reference). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviuses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. New techniques using recombinant phage antibody expression systems can also be used to generate monoclonal antibodies. See for example: McCafferty, J et al. [64]; Hoogenboom, H. R. et al. [39]; and Marks, J. D. et al. [60].

Such peptides may be produced by expressing the specific sequence in a recombinantly engineered cell such as bacteria, yeast, filamentous fungal, insect (especially employing baculoviral vectors), and mammalian cells. Those of skill in the art are knowledgeable in the numerous expression systems available for expression of herpes virus protein.

Briefly, the expression of natural or synthetic nucleic acids encoding viral protein will typically be achieved by operably linking the desired sequence or portion thereof to a promoter (which is either constitutive or inducible), and incorporated into an expression vector. The vectors are suitable for replication or integration in either prokaryotes or eukaryotes. Typical cloning vectors contain antibiotic resistance markers, genes for selection of transformants, inducible or regulatable promoter regions, and translation terminators that are useful for the expression of viral genes.

Methods for the expression of cloned genes in bacteria are also well known. In general, to obtain high level expression of a cloned gene in a prokaryotic system, it is advisable to construct expression vectors containing a strong promoter to direct mRNA transcription. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to antibiotics. See [81] supra, for details concerning selection markers and promoters for use in E. coli. Suitable eukaryote hosts may include plant cells, insect cells, mammalian cells, yeast, and filamentous fungi.

Methods for characterizing naturally processed peptides bound to MHC (major histocompatibility complex) I molecules have been developed. See, Falk et al. [24], and PCT publication No. WO 92/21033 published Nov. 26, 1992, both of which are incorporated by reference herein. Typically, these methods involve isolation of MHC class I molecules by immunoprecipitation or affinity chromatography from an appropriate cell or cell line. Other methods involve direct amino acid sequencing of the more abundant peptides in various HPLC fractions by known automatic sequencing of peptides eluted from Class I molecules of the B cell type (Jardetzkey, et al. [45], incorporated by reference herein, and of the human MHC class I molecule, HLA-A2.1 type by mass spectrometry (Hunt, et al. [40], incorporated by reference herein). See also, Rotzschke and Falk [79], incorporated by reference herein for a general review of the characterization of naturally processed peptides in MHC class I. Further, Marloes, et al. [61], incorporated by reference herein, describe how class I binding motifs can be applied to the identification of potential viral immunogenic peptides in vitro.

The peptides described herein produced by recombinant technology may be purified by standard techniques well known to those of skill in the art. Recombinantly produced viral sequences can be directly expressed or expressed as a fusion protein. The protein is then purified by a combination of cell lysis (e.g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.

The proteins may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, Scopes, R. [84], incorporated herein by reference.

B. Serological tests for the presence of antibodies to the human herpesvirus.

This invention further embraces diagnostic kits for detecting the presence of a KS agent in biological samples, such as serum or solid tissue samples, comprising a container containing antibodies to the human herpesvirus, and instructional material for performing the test. Alternatively, inactivated viral particles or peptides or viral proteins derived from the human herpesvirus may be used in a diagnostic kit to detect for antibodies specific to the KS associated human herpesvirus.

Diagnostic kits for detecting the presence of a KS agent in tissue samples, such as skin samples or samples of other affected tissue, comprising a container containing a nucleic acid sequence specific for the human herpesvirus and instructional material for detecting the KS-associated herpesvirus are also included. A container containing nucleic acid primers to any one of such sequences is optionally included as are antibodies to the human herpesvirus as described herein.

Antibodies reactive with antigens of the human herpesvirus can also be measured by a variety of immunoassay methods that are similar to the procedures described above for measurement of antigens. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see Basic and Clinical Immunology 7th Edition [12], and [32], supra.

In brief, immunoassays to measure antibodies reactive with antigens of the KS-associated human herpesvirus can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably the capture agent is a purified recombinant human herpesvirus protein produced as described above. Other sources of human herpesvirus proteins, including isolated or partially purified naturally occurring protein, may also be used. Noncompetitive assays are typically sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents. One of the binding agents is used as a capture agent and is bound to a solid surface. The second binding agent is labelled and is used to measure or detect the resultant complex by visual or instrument means. A number of combinations of capture agent and labelled binding agent can be used. A variety of different immunoassay formats, separation techniques and labels can be also be used similar to those described above for the measurement of the human herpesvirus antigens.

Hemagglutination Inhibition (HI) and Complement Fixation (CF) which are two laboratory tests that can be used to detect infection with human herpesvirus by testing for the presence of antibodies against the virus or antigens of the virus.

Serological methods can be also be useful when one wishes to detect antibody to a specific variant. For example, one may wish to see how well a vaccine recipient has responded to the new variant.

Alternatively, one may take serum from a patient to see which variant the patient responds to the best.

This invention provides an antagonist capable of blocking the expression of the peptide or polypeptide encoded by the isolated DNA molecule. In one embodiment the antagonist is capable of hybridizing with a double stranded DNA molecule. In another embodiment the antagonist is a triplex oligonucleotide capable of hybridizing to the DNA molecule. In another embodiment the triplex oligonucleotide is capable of binding to at least a portion of the isolated DNA molecule with a nucleotide sequence as shown in FIG. 3A-3F (SEQ ID NOs: 1-37).

This invention provides an antisense molecule capable of hybridizing to the isolated DNA molecule. In one embodiment the antisense molecule is DNA. In another embodiment the antisense molecule is RNA.

The antisense molecule may be DNA or RNA or variants thereof (i.e. DNA or RNA with a protein backbone). The present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the receptor recognition proteins at the translation of a specific mRNA, either by masking that MRNA with an antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific MRNA molecule. In the cell, they hybridize to that MRNA, forming a double stranded molecule. The cell does not translate an MRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of MRNA into protein. Oligomer of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon are particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules upon introduction to cells.

This invention provides a transgenic nonhuman mammal which comprises at least a portion of the isolated DNA molecule introduced into the mammal at an embryonic stage. Methods of producing a transgenic nonhuman mammal are known to those skilled in the art.

This invention provides a cell line containing the isolated KS associated herpesvirus of the subject invention. In one embodiment the isolated DNA molecule is artificially introduced into the cell. Cell lines include, but are not limited to: fibroblasts, such as HFF, NIH/3T3; Epithelial cells, such as 5637; lymphocytes, such as FCB; T-cells, such as CCRF-CEM (ATCC CCL 119); B-cells, such as BJAB and Raji (ATCC CCL 86); and myeloid cells such as K562 (ATCC CCL 243); Vero cells and carcinoma cells. Methods of producing such cell lines are known to those skilled in the art. In one embodiment the isolated KS associated herpesvirus is introduced into a RCC-1 cell line.

III. In vitro diagnostic assays for the detection of KS

This invention provides a method of diagnosing Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a tumor lesion of the subject; (b) contacting the nucleic acid molecule with a labelled nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA, under hybridizing conditions; and (c) determining the presence of the nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma in the subject.

In one embodiment the DNA molecule from the tumor lesion is amplified before step (b). In another embodiment PCR is employed to amplify the nucleic acid molecule. Methods of amplifying nucleic acid molecules are known to those skilled in the art.

A person of ordinary skill in the art will be able to obtain appropriate DNA sample for diagnosing Kaposi's sarcoma in the subject. The DNA sample obtained by the above described method may be cleaved by restriction enzyme. The uses of restriction enzymes to cleave DNA and the conditions to perform such cleavage are well-known in the art.

In the above described methods, a size fractionation may be employed which is effected by a polyacrylamide gel. In one embodiment, the size fractionation is effected by an agarose gel. Further, transferring the DNA fragments into a solid matrix may be employed before a hybridization step. One examples of such solid matrix is nitrocellulose paper.

This invention provides a method of diagnosing Kaposi's sarcoma in a subject which comprises: (a) obtaining a nucleic acid molecule from a suitable bodily fluid of the subject; (b) contacting the nucleic acid molecule with a labelled nucleic acid molecules of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA, under hybridizing conditions; and (c) determining the presence of the nucleic acid molecule hybridized, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma in the subject.

This invention provides a method of diagnosing a DNA virus in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, (b) contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antibody, so as to bind the Kaposi's sarcoma antibody to a specific Kaposi's sarcoma antigen, (c) removing unbound bodily fluid from the support, and (d) determining the level of Kaposi's sarcoma antibody bound by the Kaposi's sarcoma antigen, thereby diagnosing the subject for Kaposi's sarcoma.

This invention provides a method of diagnosing Kaposi's sarcoma in a subject, which comprises (a) obtaining a suitable bodily fluid sample from the subject, (b) contacting the suitable bodily fluid of the subject to a support having already bound thereto a Kaposi's sarcoma antigen, so as to bind Kaposi's sarcoma antigen to a specific Kaposi's sarcoma antibody, (c) removing unbound bodily fluid from the support, and (d) determining the level of the Kaposi's sarcoma antigen bound by the Kaposi's sarcoma antibody, thereby diagnosing Kaposi's sarcoma.

This invention provides a method of detecting expression of a DNA virus associated with Kaposi's sarcoma in a cell which comprises obtaining total cDNA obtained from the cell, contacting the cDNA so obtained with a labelled DNA molecule under hybridizing conditions, determining the presence of cDNA hybridized to the molecule, and thereby detecting the expression of the DNA virus. In one embodiment mRNA is obtained from the cell to detect expression of the DNA virus.

The suitable bodily fluid sample is any bodily fluid sample which would contain Kaposi's sarcoma antibody, antigen or fragments thereof. A suitable bodily fluid includes, but is not limited to: serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, or exudates. In the preferred embodiment, the suitable bodily fluid sample is serum or plasma. In addition, the bodily fluid sample may be cells from bone marrow, or a supernatant from a cell culture. Methods of obtaining a suitable bodily fluid sample from a subject are known to those skilled in the art. Methods of determining the level of antibody or antigen include, but are not limited to: ELISA, IFA, and Western blotting. Other methods are known to those skilled in the art. Further, a subject infected with a DNA virus associated with Kaposi's sarcoma may be diagnosed with the above described methods.

The detection of the human herpesvirus and the detection of virus-associated KS are essentially identical processes. The basic principle is to detect the virus using specific ligands that bind to the virus but not to other proteins or nucleic acids in a normal human cell or its environs. The ligands can either be nucleic acid or antibodies. The ligands can be naturally occurring or genetically or physically modified such as nucleic acids with non-natural or antibody derivatives, i.e., Fab or chimeric antibodies. Serological tests for detection of antibodies to the virus may also be performed by using protein antigens obtained from the human herpesvirus, and described herein.

Samples can be taken from patients with KS or from patients at risk for KS, such as AIDS patients. Typically the samples are taken from blood (cells, serum and/or plasma) or from solid tissue samples such as skin lesions. The most accurate diagnosis for KS will occur if elevated titers of the virus are detected in the blood or in involved lesions. KS may also be indicated if antibodies to the virus are detected and if other diagnostic factors for KS is present.

A. Nucleic acid assays.

The diagnostic assays of the invention can be nucleic acid assays such as nucleic acid hybridization assays and assays which detect amplification of specific nucleic acid to detect for a nucleic acid sequence of the human herpesvirus described herein.

Accepted means for conducting hybridization assays are known and general overviews of the technology can be had from a review of: Nucleic Acid Hybridization: A Practical Approach [72]; Hybridization of Nucleic Acids Immobilized on Solid Supports [41]; Analytical Biochemistry [4] and Innis et al., PCR Protccols [74], supra, all of which are incorporated by reference herein.

If PCR is used in conjunction with nucleic acid hybridization, primers are designed to target a specific portion of the nucleic acid of the herpesvirus. For example, the primers set forth in SEQ ID NOs: 38-40 may be used to target detection of regions of the herpesvirus genome encoding ORF 25 homologue--ORF 32 homologue. From the information provided herein, those of skill in the art will be able to select appropriate specific primers.

Target specific probes may be used in the nucleic acid hybridization diagnostic assays for KS. The probes are specific for or complementary to the target of interest. For precise allelic differentiations, the probes should be about 14 nucleotides long and preferably about 20-30 nucleotides. For more general detection of the human herpesvirus of the invention, nucleic acid probes are about 50 to about 1000 nucleotides, most preferably about 200 to about 400 nucleotides.

A sequence is "specific" for a target organism of interest if it includes a nucleic acid sequence which when detected is determinative of the presence of the organism in the presence of a heterogeneous population of proteins and other biologics. A specific nucleic acid probe is targeted to that portion of the sequence which is determinative of the organism and will not hybridize to other sequences especially those of the host where a pathogen is being detected.

The specific nucleic acid probe can be RNA or DNA polynucleotide or oligonucleotide, or their analogs. The probes may be single or double stranded nucleotides. The probes of the invention may be synthesized enzymatically, using methods well known in the art (e.g., nick translation, primer extension, reverse transcription, the polymerase chain reaction, and others) or chemically (e.g., by methods such as the phosphoramidite method described by Beaucage and Carruthers [19], or by the triester method according to Matteucci, et al. [62], both incorporated herein by reference).

The probe must be of sufficient length to be able to form a stable duplex with its target nucleic acid in the sample, i.e., at least about 14 nucleotides, and may be longer (e.g., at least about 50 or 10) bases in length). Often the probe will be more than about 100 bases in length. For example, when probe is prepared by nick-translation of DNA in the presence of labeled nucleotides the average probe length may be about 100-600 bases.

As noted above, the probe will be capable of specific hybridization to a specific KS-associated herpes virus nucleic acid. Such "specific hybridization" occurs when a probe hybridizes to a target nucleic acid, as evidenced by a detectable signal, under conditions in which the probe does not hybridize to other nucleic acids (e.g., animal cell or other bacterial nucleic acids) present in the sample. A variety of factors including the length and base composition of the probe, the extent of base mismatching between the probe and the target nucleic acid, the presence of salt and organic solvents, probe concentration, and the temperature affect hybridization, and optimal hybridization conditions must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, [81], supra, Ausubel, F., et al. [8] [hereinafter referred to as Sambrook], Methods in Enzymology [67] or Hybridization with Nucleic Acid Probes [42] all of which are incorporated herein by reference.

Usually, at least a part of the probe will have considerable sequence identity with the target nucleic acid. Although the extent of the sequence identity required for specific hybridization will depend on the length of the probe and the hybridization conditions, the probe will usually have at least 70% identity to the target nucleic acid, more usually at least 80% identity, still more usually at least 90% identity and most usually at least 95% or 100% identity.

A probe can be identified as capable of hybridizing specifically to its target nucleic acid by hybridizing the probe to a sample treated according the protocol of this invention where the sample contains both target virus and animal cells (e.g., nerve cells). A probe is specific if the probe's characteristic signal is associated with the herpesvirus DNA in the sample and not generally with the DNA of the host cells and non-biological materials (e.g., substrate) in a sample.

The following stringent hybridization and washing conditions will be adequate to distinguish a specific probe (e.g., a fluorescently labeled DNA probe) from a probe that is not specific: incubation of the probe with the sample for 12 hours at 37° C. in a solution containing denatured probe, 50% formamide, 2× SSC, and 0.1% (w/v) dextran sulfate, followed by washing in 1× SSC at 70° C. for 5 minutes; 2× SSC at 37° C. for 5 minutes; 0.2× SSC at room temperature for 5 minutes, and H₂ O at room temperature for 5 minutes. Those of skill will be aware that it will often be advantageous in nucleic acid hybridizations (i.e., in situ, Southern, or other) to include detergents (e.g., sodium dodecyl sulfate), chelating agents (e.g., EDTA) or other reagents (e.g., buffers, Denhardt's solution, dextran sulfate) in the hybridization or wash solutions. To test the specificity of the virus specific probes, the probes can be tested on host cells containing the KS-associated herpesvirus and compared with the results from cells containing non-KS-associated virus.

It will be apparent to those of ordinary skill in the art that a convenient method for determining whether a probe is specific for a KS-associated viral nucleic acid utilizes a Southern blot (or Dot blot) using DNA prepared from one or more KS-associated human herpesviruses of the invention. Briefly, to identify a target specific probe DNA is isolated from the virus. Test DNA either viral or cellular is transferred to a solid (e.g., charged nylon) matrix. The probes are labelled following conventional methods. Following denaturation and/or prehybridization steps known in the art, the probe is hybridized to the immobilized DNAs under stringent conditions. Stringent hybridization conditions will depend on the probe used and can be estimated from the calculated T_(m) (melting temperature) of the hybridized probe (see, e.g., Sambrook for a description of calculation of the T_(m)). For radioactively-labeled DNA or RNA probes an example of stringent hybridization conditions is hybridization in a solution containing denatured probe and 5× SSC at 65° C. for 8-24 hours followed by washes in 0.1× SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65° C. In general, the temperature and salt concentration are chosen so that the post hybridization wash occurs at a temperature that is about 5° C. below the T_(M) of the hybrid. Thus for a particular salt concentration the temperature may be selected that is 5° C. below the T_(M) or conversely, for a particular temperature, the salt concentration is chosen to provide a T_(M) for the hybrid that is 5° C. warmer than the wash temperature. Following stringent hybridization and washing, a probe that hybridizes to the KS-associated viral DNA but not to the non-KS associated viral DNA, as evidenced by the presence of a signal associated with the appropriate target and the absence of a signal from the non-target nucleic acids, is identified as specific for the KS associated virus. It is further appreciated that in determining probe specificity and in utilizing the method of this invention to detect KS-associated herpesvirus, a certain amount of background signal is typical and can easily be distinguished by one of skill from a specific signal. Two fold signal over background is acceptable.

A preferred method for detecting the KS-associated herpesvirus is the use of PCR and/or dot blot hybridization. The presence or absence of an KS agent for detection or prognosis, or risk assessment for KS includes Southern transfers, solution hybridization or non-radioactive detection systems, all of which are well known to those of skill in the art. Hybridization is carried out using probes. Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the causal agent.

Similarly, a Northern transfer may be used for the detection of message in samples of RNA or reverse transcriptase PCR and cDNA can be detected by methods described above. This procedure is also well known in the art. See [81] incorporated by reference herein.

An alternative means for determining the presence of the human herpesvirus is in situ hybridization, or more recently, in situ polymerase chain reaction. In situ PCR is described in Neuvo et al. [71], Intracellular localization of polymerase chain reaction (PCR)-amplified Hepatitis C cDNA; Bagasra et al. [10], Detection of Human Immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction; and Heniford et al. [35], Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. In situ hybridization assays are well known and are generally described in Methods Enzymol. [67] incorporated by reference herein. In an in situ hybridization, cells are fixed to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of target-specific probes that are labelled. The probes are preferably labelled with radioisotopes or fluorescent reporters.

The above described probes are also useful for in-situ hybridization or in order to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its MRNA in various biological tissues. In-situ hybridization is a sensitive localization method which is not dependent on expression of antigens or native vs. denatured conditions.

Oligonucleotide (oligo) probes, synthetic oligonucleotide probes or riboprobes made from KSHV phagemids/plasmids, are relatively homogeneous reagents and successful hybridization conditions in tissue sections is readily transferable from one probe to another. Commercially synthesized oligonucleotide probes are prepared against the identified genes. These probes are chosen for length (45-65 mers), high G-C content (50-70%) and are screened for uniqueness against other viral sequences in GenBank.

Oligonucleotides are 3'end-labeled with [α-³⁵ S]dATP to specific activities in the range of 1×10¹⁰ dpm/ug using terminal deoxynucleotidyl transferase. Unincorporated labeled nucleotides are removed from the oligo probe by centrifugation through a Sephadex G-25 column or by elution from a Waters Sep Pak C-18 column.

KS tissue embedded in OCT compound and snap frozen in freezing isopentane cooled with dry ice is cut at 6 μm intervals and thawed onto 3-aminopropyltriethoxysilane treated slides and allowed to air dry. The slides are then be fixed in 4% freshly prepared paraformaldehyde, rinsed in water. Formalin-fixed, paraffin embedded KS tissues cut at 6 μm and baked onto glass slides can also be used. The sections are then deparaffinized in xylenes and rehydrated through graded alcohols. Prehybridization in 20 mM Tris Ph 7.5, 0.02% Denhardt's solution, 10% dextran sulfate for 30 min at 37° C. is followed by hybridization overnight in a solution of 50% formamide (v/v), 10% dextran sulfate (w/v), 20 mM sodium phosphate (Ph 7.4), 3× SSC, 1× Denhardt's solution, 100 ug/ml salmon sperm DNA, 125 ug/ml yeast tRNA and the oligo probe (10⁶ cpm/ml) at 42° C. overnight. The slides are washed twice with 2× SSC and twice with 1× SSC for 15 minutes each at room temperature and visualized by autoradiography. Briefly, sections are dehydrated through graded alcohols containing 0.3M ammonium acetate and air dried. The slides are dipped in Kodak NTB2 emulsion, exposed for days to weeks, developed, and counterstained with hematoxylin and eoxin. Alternative immunohistochemical protocols may be employed which are known to those skilled in the art.

IV. Treatment of human herpesvirus-induced KS

This invention provides a method of treating a subject with Kaposi's sarcoma, comprising administering to the subject an effective amount of the antisense molecule capable of hybridizing to the isolated DNA molecule under conditions such that the antisense molecule selectively enters a tumor cell of the subject, so as to treat the subject.

This invention provides a method for treating a subject with Kaposi's sarcoma (KS) comprising administering to the subject having a human herpesvirus-associated KS a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier, wherein the agent is effective to treat the subject with KS-associated human herpes virus.

Further, this invention provides a method of prophylaxis or treatment for Kaposi's sarcoma (KS) by administering to a patient at risk for KS, an antibody that binds to the human herpesvirus in a pharmaceutically acceptable carrier. In one embodiment the antiviral drug is used to treat a subject with the DNA herpesvirus of the subject invention.

The use of combinations of antiviral drugs and sequential treatments are useful for treatment of herpesvirus infections and will also be useful for the treatment of herpesvirus-induced KS. For example, Snoeck et al. [88], found additive or synergistic effects against CMV when combining antiherpes drugs (e.g., combinations of zidovudine [3'-azido-3'-deoxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals). Similarly, in treatment of cytomegalovirus retinitis, induction with ganciclovir followed by maintenance with foscarnet has been suggested as a way to maximize efficacy while minimizing the adverse side effects of either treatment alone. An anti-herpetic composition that contains acyclovir and, e.g., 2-acetylpyridine-5-((2-pyridylamino)thiocarbonyl)-thiocarbonohydrazone is described in U.S. Pat. No. 5,175,165 (assigned to Burroughs Wellcome Co.). Combinations of TS-inhibitors and viral TK-inhibitors in antiherpetic medicines are disclosed in U.S. Pat. No. 5,137,724, assigned to Stichting Rega VZW. A synergistic inhibitory effect on EBV replication using certain ratios of combinations of HPMPC with AZT was reported by Lin et al. [56].

U.S. Pat. Nos. 5,164,395 and 5,021,437 (Blumenkopf; Burroughs Wellcome) describe the use of a ribonucleotide reductase inhibitor (an acetylpyridine derivative) for treatment of herpes infections, including the use of the acetylpyridine derivative in combination with acyclovir. U.S. Pat. No. 5,137,724 (Balzari et al. [11]) describes the use of thymilydate synthase inhibitors (e.g., 5-fluoro-uracil and 5-fluro-2'-deoxyuridine) in combination with compounds having viral thymidine kinase inhibiting activity.

With the discovery of a disease causal agent for KS now identified, effective therapeutic or prophalactic protocols to alleviate or prevent the symptoms of herpes virus-associated KS can be formulated. Due to the viral nature of the disease, antiviral agents have application here for treatment, such as interferons, nucleoside analogues, ribavirin, amantadine, and pyrophosphate analogues of phosphonoacetic acid (foscarnet) (reviewed in Gorbach, S. L., et al. [28]) and the like. Immunological therapy will also be effective in many cases to manage and alleviate symptoms caused by the disease agents described here. Antiviral agents include agents or compositions that directly bind to viral products and interfere with disease progress; and, excludes agents that do not impact directly on viral multiplication or viral titer. Antiviral agents do not include immunoregulatory agents that do not directly affect viral titer or bind to viral products. Antiviral agents are effective if they inactivate the virus, otherwise inhibit its infectivity or multiplication, or alleviate the symptoms of KS.

A. Antiviral Agents.

The antiherpesvirus agents that will be useful for treating virus-induced KS can be grouped into broad classes based on their presumed modes of action. These classes include agents that act (i) by inhibition of viral DNA polymerase, (ii) by targeting other viral enzymes and proteins, (iii) by miscellaneous or incompletely understood mechanisms, or (iv) by binding a target nucleic acid (i.e., inhibitory nucleic acid therapeutics). Antiviral agents may also be used in combination (i.e. together or sequentially) to achieve synergistic or additive effects or other benefits.

Although it is convenient to group antiviral agents by their supposed mechanism of action, the applicants do not intend to be bound by any particular mechanism of antiviral action. Moreover, it will be understood by those of skill that an agent may act on more than one target in a virus or virus-infected cell or through more than one mechanism.

i) Inhibitors of viral DNA polymerase

Many antiherpesvirus agents in clinical use or in development today are nucleoside analogs believed to act through inhibition of viral DNA replication, especially through inhibition of viral DNA polymerase. These nucleoside analogs act as alternative substrates for the viral DNA polymerase or as competitive inhibitors of DNA polymerase substrates. Usually these agents are preferentially phosphorylated by viral thymidine kinase (TK), if one is present, and/or have higher affinity for viral DNA polymerase than for the cellular DNA polymerases, resulting in selective antiviral activity. Where a nucleoside aralogue is incorporated into the viral DNA, viral activity or reproduction may be affected in a variety of ways. For example, the analogue may act as a chain terminator, cause increased lability (e.g., susceptibility to breakage) of analogue-containing DNA, and/or impair the ability of the substituted DNA to act as template for transcription or replication (see, e.g., Balzarini et al. [11]).

It will be known to one of skill that, like many drugs, many of the agents useful for treatment of herpes virus infections are modified (i.e., "activated") by the host, host cell, or virus-infected host cell metabolic enzymes. For example, acyclovir is triphosphorylated to its active form, with the first phosphorylation being carried out by the herpes virus thymidine kinase, when present. Other examples are the reported conversion of the compound HOE 602 to ganciclovir in a three-step metabolic pathway (Winkler et al. [95]) and the phosphorylation of ganciclovir to its active form by, e.g., a CMV nucleotide kinase. It will be apparent to one of skill that the specific metabolic capabilities of a virus can affect the sensitivity of that virus to specific drugs, and is one factor in the choice of an antiviral drug. The mechanism of action of certain anti-herpesvirus agents is discussed in De Clercq [22] and in other references cited supra and infra, all of which are incorporated by reference herein.

Anti-herpesvirus medications suitable for treating viral induced KS include, but are not limited to, nucleoside analogs including acyclic nucleoside phosphonate analogs (e.g., phosphonylmethoxyalkylpurines and -pyrimidines), and cyclic nucleoside analogs. These include drugs such as: vidarabine (9-β-D-arabinofuranosyladenine; adenine arabinoside, ara-A, Vira-A, Parke-Davis); 1-β-D-arabinofuranosyluracil (ara-U); 1-β-D-arabinofuranosyl-cytosine (ara-C) ; HPMPC [(S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl] cytosine (e.g., GS 504 Gilead Science) and its cyclic form (cHPMPC); HPMPA [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] and its cyclic form (cHPMPA); (S)-HPMPDAP [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine]; PMEDAP [9-(2-phosphonyl-methoxyethyl)-2,6-diaminopurine]; HOE 602 [2-amino-9-(1,3-bis(isopropoxy)-2-propoxymethyl)purine]; PMEA [9-(2-phosphonylmethoxyethyl)adenine]; bromovinyl-deoxyuridine (Burns and Sandford. [21]); 1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)-uridine or -2'-deoxyuridine; BVaraU (1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)-uracil, brovavir, Bristol-Myers Squibb, Yamsa Shoyu); BVDU [(E)-5-(2-bromovinyl)-2'-deoxyuridine, brivudin, e.g., Helpin] and its carbocyclic analogue (in which the sugar moiety is replaced by a cyclopentane ring); IVDU [(E)-5-(2-iodovinyl)-2'-deoxyuridine] and its carbocyclic analogue, C-IVDU (Balzarini et al. [11])]; and 5-mercutithio analogs of 2'-deoxyuridine (Holliday, J., and Williams, M. V. [38]); acyclovir [9-([2-hydroxyethoxy]methyl)guanine; e.g., Zovirax (Burroughs Wellcome)]; penciclovir (9-[4-hydroxy-2-(hydroxymethyl)butyl]-guanine); ganciclovir [(9-[1,3-dihydroxy-2 propoxymethyl]-guanine) e.g., Cymevene, Cytovene (Syntex), DHPG (Stals et al. [89]]; isopropylether derivatives of ganciclovir (see, e.g., Winkelmann et al. [94]); cygalovir; famciclovir [2-amino-9-(4-acetoxy-3-(acetoxymethyl)but-1-yl)purine (Smithkline Beecham)]; valacyclovir (Burroughs Wellcome); desciclovir [(2-amino-9-(2-ethoxymethyl)purine)] and 2-amino-9-(2-hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG (carbocyclic 2'-deoxyguanosine); and purine nucleosides with the pentafuranosyl ring replaced by a cyclo butane ring (e.g., cyclobut-A [(+-)-9-[1β,2α,3β)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine], cyclobut-G [(+-)-9-[1β,2α,3β)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine], BHCG [(R)-(1α,2β,1α)-9-(2,3-bis(hydroxymethyl)cyclobutyl]guanine], and an active isomer of racemic BHCG, SQ 34,514 [1R-1α,2β,3α)-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one (see, Braitman et al.(1991) [20]]. Certain of these antiherpesviral agents are discussed in Gorach et al. [28]; Saunders et al. [82]; Yamanaka et al., [96]; Greenspan et al. [29], all of which are incorporated by reference herein.

Triciribine and triciribine monophosphate are potent inhibitors against herpes viruses. (Ickes et al. [43], incorporated by reference herein), HIV-1 and HIV-2 (Kucera et al. [51], incorporated by reference herein) and are additional nucleoside analogs that may be used to treat KS. An exemplary protocol for these agents is an intravenous injection of about 0.35 mg/meter² (0.7 mg/kg) once weekly or every other week for at least two doses, preferably up to about four to eight weeks.

Acyclovir and ganciclovir are of interest because of their accepted use in clinical settings. Acyclovir, an acyclic analogue of guanine, is phosphorylated by a herpesvirus thymidine kinase and undergoes further phosphorylation to be incorporated as a chain terminator by the viral DNA polymerase during viral replication. It has therapeutic activity against a broad range of herpesviruses, Herpes simplex Types 1 and 2, Varicella- Zoster, Cytomegalovirus, and Epstein-Barr Virus, and is used to treat disease such as herpes encephalitis, neonatal herpesvirus infections, chickenpox in immunocompromised hosts, herpes zoster recurrences, CMV retinitis, EBV infections, chronic fatigue syndrome, and hairy leukoplakia in AIDS patients. Exemplary intravenous dosages or oral dosages are 250 mg/kg/m² body surface area, every 8 hours for 7 days, or maintenance doses of 200-400 mg IV or orally twice a day to suppress recurrence. Ganciclovir has been shown to be more active than acyclovir against some herpesviruses. See, e.g., Oren and Soble [73]. Treatment protocols for ganciclovir are 5 mg/kg twice a day IV or 2.5 mg/kg three times a day for 10-14 days. Maintenance doses are 5-6 mg/kg for 5-7 days.

Also of interest is HPMPC. HPMPC is reported to be more active than either acyclovir or ganciclovir in the chemotherapy and prophylaxis of various HSV-1, HSV-2, TK- HSV, VZV or CMV infections in animal models ([22], supra).

Nucleoside analogs such as BVaraU are potent inhibitors of HSV-1, EBV, and VZV that have greater activity than acyclovir in animal models of encephalitis. FIAC (fluroidoarbinosyl cytosine) and its related fluroethyl and iodo compounds (e.g., FEAU, FIAU) have potent selective activity against herpesviruses, and HPMPA ((S)-1-([3-hydroxy-2-phosphorylmethoxy]propyl)adenine) has been demonstrated to be more potent against HSV and CMV than acyclovir or ganciclovir and are of choice in advanced cases of KS. Cladribine (2-chlorodeoxyadenosine) is another nucleoside analogue known as a highly specific antilymphocyte agent (i.e., a immunosuppressive drug).

Other useful antiviral agents include: 5-thien-2-yl-2'-deoxyuridine derivatives, e.g., BTDU [5-5(5-bromothien-2-yl)-2'-deoxyuridine] and CTDU [b-(5-chlorothien-2-yl)-2'-deoxyuridine]; and OXT-A [9-(2-deoxy-2-hydroxymethyl-β-D-erythro-oxetanosyl)adenine] and OXT-G [9-(2-deoxy-2-hydroxymethyl-β-D-erythro-oxetanosyl)guanine]. Although OXT-G is believed to act by inhibiting viral DNA synthesis its mechanism of action has not yet been elucidated. These and other compounds are described in Andrei et al. [5] which is incorporated by reference herein. Additional antiviral purine derivatives useful in treating herpesvirus infections are disclosed in U.S. Pat. No. 5,108,994 (assigned to Beecham Group P.L.C.). 6-Methoxypurine arabinoside (ara-M; Burroughs Wellcome) is a potent inhibitor of varicella-zoster virus, and will be useful for treatment of KS.

Certain thymidine analogs [e.g., idoxuridine (5-ido-2'-deoxyuridine)] and triflurothymidine) have antiherpes viral activity, but due to their systemic toxicity, are largely used for topical herpesviral infections, including HSV stromal keratitis and uveitis, and are not preferred here unless other options are ruled out.

Other useful antiviral agents that have demonstrated antiherpes viral activity include foscarnet sodium (trisodium phosphonoformate, PFA, Foscavir (Astra)) and phosphonoacetic acid (PAA). Foscarnet is an inorganic pyrophosphate analogue that acts by competitively blocking the pyrophosphate-binding site of DNA polymerase. These agents which block DNA polymerase directly without processing by viral thymidine kinase. Foscarnet is reported to be less toxic than PAA.

ii) Agents that target viral proteins other than DNA polymerase or other viral functions.

Although applicants do not intend to be bound by a particular mechanism of antiviral action, the antiherpes-virus agents described above are believed to act through inhibition of viral DNA polymerase. However, viral replication requires not only the replication of the viral nucleic acid but also the production of viral proteins and other essential components. Accordingly, the present invention contemplates treatment of KS by the inhibition of viral proliferation by targeting viral proteins other than DNA polymerase (e.g., by inhibition of their synthesis or activity, or destruction of viral proteins after their synthesis). For example, administration of agents that inhibit a viral serine protease, e.g., such as one important in development of the viral capsid will be useful in treatment of viral induced KS.

Other viral enzyme targets include: OMP decarboxylase inhibitors (a target of, e.g., parazofurin), CTP synthetase inhibitors (targets of, e.g., cyclopentenylcytosine), IMP dehydrogenase, ribonucleotide reductase (a target of, e.g., carboxyl-containing N-alkyldipeptides as described in U.S. Pat. No. 5,110,799 (Tolman et al., Merck)), thymidine kinase (a target of, e.g., 1-[2-(hydroxymethyl)cycloalkylmethyl]-5-substituted -uracils and -guanines as described in, e.g., U.S. Pat. Nos. 4,863,927 and 4,782,062 (Tolman et al.; Merck)) as well as other enzymes. It will be apparent to one of ordinary skill in the art that there are additional viral proteins, both characterized and as yet to be discovered, that can serve as target for antiviral agents.

iv) Other agents and modes of antiviral action.

Kutapressin is a liver derivative available from Schwarz Parma of Milwaukee, Wis. in an injectable form of 25 mg/ml. The recommended dosage for herpesviruses is from 200 to 25 mg/ml per clay for an average adult of 150 pounds.

Poly(I) Poly(C₁₂ U), an accepted antiviral drug known as Ampligen from HEM Pharmaceuticals of Rockville, Md. has been shown to inhibit herpesviruses and is another antiviral agent suitable for treating KS. Intravenous injection is the preferred route of administration. Dosages from about 100 to 600 mg/m² are administered two to three times weekly to adults averaging 150 pounds. It is best to administer at least 200 mg/m² per week.

Other antiviral agents reported to show activity against herpes viruses (e.g., varicella zoster and herpes simplex) and will be useful for the treatment of herpesvirus-induced KS include mappicine ketone (SmithKline Beecham); Compounds A,79296 and A,73209 (Abbott) for varicella zoster, and Compound 882C87 (Burroughs Wellcome) [see, The Pink Sheet 55(20) May 17, 1993].

Interferon is known inhibit replication of herpes viruses. See [73], supra. Interferon has known toxicity problems and it is expected that second generation derivatives will soon be available that will retain interferon's antiviral properties but have reduced side affects.

It is also contemplated that herpes virus-induced KS may be treated by administering a herpesvirus reactivating agent to induce reactivation of the latent virus. Preferably the reactivation is combined with simultaneous or sequential administration of an anti-herpesvirus agent. Controlled reactivation over a short period of time or reactivation in the presence of an antiviral agent is believed to minimize the adverse effects of certain herpesvirus infections (e.g., as discussed in PCT Application WO 93/04683). Reactivating agents include agents such as estrogen, phorbol esters, forskolin and β-adrenergic blocking agents.

Agents useful for treatment of herpesvirus infections and for treatment of herpesvirus-induced KS are described in numerous U.S. Patents. For example, ganciclovir is an example of a antiviral guanine acyclic nucleotide of the type described in U.S. Pat. Nos. 4,355,032 and 4,603,219.

Acyclovir is an example of a class of antiviral purine derivatives, including 9-(2-hydroxyethylmethyl)adenine, of the type described in U.S. Pat. Nos. 4,287,188, 4,294,831 and 4,199,574.

Brivudin is an example of an antiviral deoxyuridine derivative of the type described in U.S. Pat. No. 4,424,211.

Vidarabine is an example of an antiviral purine nucleoside of the type described in British Pat. 1,159,290.

Brovavir is an example of an antiviral deoxyuridine derivative of the type described in U.S. Pat. Nos. 4,542,210 and 4,386,076.

BHCG is an example of an antiviral carbocyclic nucleoside analogue of the type described in U.S. Pat. Nos. 5,153,352, 5,034,394 and 5,126,345.

HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivative with of the type described in U.S. Pat. No. 5,142,051.

CDG (Carbocyclic 2'-deoxyguanosine) is an example of an antiviral carbocyclic nucleoside analogue of the type described in U.S. Pat. Nos. 4,543,255, 4,855,466, and 4,894,458.

Foscarnet is described in U.S. Pat. No. 4,339,445.

Trifluridine and its corresponding ribonucleoside is described in U.S. Pat. No. 3,201,387.

U.S. Pat. No. 5,321,030 (Kaddurah-Daouk et al.; Amira) describes the use of creatine analogs as antiherpes viral agents. U.S. Pat. No. 5,306,722 (Kim et al.; Bristol-Meyers Squibb) describes thymidine kinase inhibitors useful for treating HSV infections and for inhibiting herpes thymidine kinase. Other antiherpesvirus compositions are described in U.S. Pat. Nos. 5,286,649 and 5,098,708 (Konishi et al., Bristol-Meyers Squibb) and 5,175,165 (Blumenkopf et al.; Burroughs Wellcome). U.S. Pat. No. 4,880,820 (Ashton et al.; Merck) describes the antiherpes virus agent (S)-9-(2,3-dihydroxy-1-propoxymethyl)guanine.

U.S. Pat. No. 4,708,935 (Suhadolnik et al.; Research Corporation) describes a 3'-deoxyadenosine compound effective in inhibiting HSV and EBV. U.S. Pat. No. 4,386,076 (Machida et al.; Yamasa Shoyu Kabushiki Kaisha) describes use of (E)-5-(2-halogenovinyl)-arabinofuranosyluracil as an antiherpesvirus agent. U.S. Pat. No. 4,340,599 (Lieb et al.; Bayer Aktiengesellschaft) describes phosphonohydroxyacetic acid derivatives useful as antiherpes agents. U.S. Pat. Nos. 4,093,715 and 4,093,716 (Lin et al. Research Corporation) describe 5'-amino-5'-deoxythymidine and 5-iodo-5'-amino-2',5'-dideoxycytidine as potent inhibitors of herpes simplex virus. U.S. Pat. No. 4,069,382 (Baker et al.; Parke, Davis & Company) describes 9-(5-O-Acyl-beta-D-arabinofuranosyl)adenine compounds useful as antiviral agents. U.S. Pat. No. 3,927,216 (Witkowski et al.) describes the use of 1,2,4-triazole-3-carboxamide and 1,2,4-triazole-3-thiocarboxamide for inhibiting herpes virus infections. U.S. Pat. No. 5,179,093 (Afonso et al., Schering) describes quinoline-2,4-dione derivatives active against herpes simplex virus 1 and 2, cytomegalovirus and Epstein Barr virus.

v) Inhibitory nucleic acid therapeutics

Also contemplated here are inhibitory nucleic acid therapeutics which can inhibit the activity of herpesviruses in patients with KS. Inhibitory nucleic acids may be single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex is formed. These nucleic acids are often termed "antisense" because they are usually complementary to the sense or coding strand of the gene, although recently approaches for use of "sense" nucleic acids have also been developed. The term "inhibitory nucleic acids" as used herein, refers to both "sense" and "antisense" nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking DNA transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation. Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of herpesvirus genes. These different types of inhibitory nucleic acid technology are described in Helene, C. and Toulme, J. [34], which is hereby incorporated by reference and is referred to hereinafter as "Helene and Toulme."

In brief, inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre-mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids.

Approaches targeting DNA fall into several categories. Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription. See Helene and Toulme.

More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation.

The inhibitory nucleic acids can be targeted to mRNA. In this approach, the inhibitory nucleic acids are designed to specifically block translation of the encoded protein. Using this approach, the Inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of translation of mRNA encoding critical proteins. For example, an inhibitory nucleic acid complementary to regions of c-myc mRNA inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which overexpresses the c-myc proto-oncogene. See Wickstrom E. L., et al. [93] and Harel-Bellan, A., et al. [31A]. As described in Helene and Toulme, inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms to inhibit translation of the encoded protein(s).

The inhibitory nucleic acids introduced into the cell can also encompass the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation. See Helene and Toulme.

Lastly, the inhibitory nucleic acids can be used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell. Other chemical modifications of the target nucleic acids induced by appropriately derivatized inhibitory nucleic acids may also be used.

Cleavage, and therefore inactivation, of the target nucleic acids may be effected by attaching a substituent to the inhibitory nucleic acid which can be activated to induce cleavage reactions. The substituent can be one that affects either chemical, or enzymatic cleavage. Alternatively, cleavage can be induced by the use of ribozymes or catalytic RNA. In this approach, the inhibitory nucleic acids would comprise either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.

The targeting of inhibitory nucleic acids to specific cells of the immune system by conjugation with targeting moieties binding receptors on the surface of these cells can be used for all of the above forms of inhibitory nucleic acid therapy. This invention encompasses all of the forms of inhibitory nucleic acid therapy as described above and as described in Helene and Toulme.

This invention relates to the targeting of inhibitory nucleic acids to sequences the human herpesvirus of the invention for use in treating KS. An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922 (ISIS Pharmaceuticals) which has activity against CMV [see, Biotechnology News 14 (14) p. 5].

A problem associated with inhibitory nucleic acid therapy is the effective delivery of the inhibitory nucleic acid to the target cell in vivo and the subsequent internalization of the inhibitory nucleic acid by that cell. This can be accomplished by linking the inhibitory nucleic acid to a targeting moiety to form a conjugate that binds to a specific receptor on the surface of the target infected cell, and which is internalized after binding.

iii) Administration

The subjects to be treated or whose tissue nay be used herein may be a mammal, or more specifically a human, horse, pig, rabbit, dog, monkey, or rodent. In the preferred embodiment the subject is a human.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each subject.

Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.

As used herein administration means a method of administering to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, administration topically, parenterally, orally, intravenously, intramuscularly, subcutaneously or by aerosol. Administration of the agent may be effected continuously or intermittently such that the therapeutic agent in the patient is effective to treat a subject with Kaposi's sarcoma or a subject infected with a DNA virus associated with Kaposi's sarcoma.

The antiviral compositions for treating herpesvirus-induced KS are preferably administered to human patients via oral, intravenous or parenteral administrations and other systemic forms. Those of skill in the art will understand appropriate administration protocol for the individual compositions to be employed by the physician.

The pharmaceutical formulations or compositions of this invention may be in the dosage form of solid, semi-solid, or liquid such as, e.g., suspensions, aerosols or the like. Preferably the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants; or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Effective amounts of such diluent or carrier are those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, or biological activity, etc.

V. Immunological Approaches to Therapy.

Having identified a primary causal agent of KS in humans as a novel human herpesvirus, there are immunosuppressive therapies that can modulate the immunologic dysfunction that arises from the presence of viral infected tissue. In particular, agents that block the immunological attack of the viral infected cells will ameliorate the symptoms of KS and/or reduce the disease progress. Such therapies include antibodies that specifically block the targeting of viral infected cells. Such agents include antibodies which bind to cytokines that upregulate the immune system to target viral infected cells.

The antibody may be administered to a patient either singly or in a cocktail containing two or more antibodies, other therapeutic agents, compositions, or the like, including, but not limited to, immuno-suppressive agents, potentiators and side-effect relieving agents. Of particular interest are immuno-suppressive agents useful in suppressing allergic reactions of a host. Immunosuppressive agents of interest include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, Pa.), cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination. Potentiators of interest include monensin, ammonium chloride and chloroquine. All of these agents are administered in generally accepted efficacious dose ranges such as those disclosed in the Physician Desk Reference, 41st Ed. (1987), Publisher Edward R. Barnhart, New Jersey.

Immune globulin from persons previously infected with human herpesviruses or related viruses can be obtained using standard techniques. Appropriate titers of antibodies are known for this therapy and are readily applied to the treatment of KS. Immune globulin can be administered via parenteral injection or by intrathecal shunt. In brief, immune globulin preparations may be obtained from individual donors who are screened for antibodies to the KS-associated human herpesvirus, and plasmas from high-titered donors are pooled. Alternatively, plasmas from donors are pooled and then tested for antibodies to the human herpesvirus of the invention; high-titered pools are then selected for use in KS patients.

Antibodies may be formulated into an injectable preparation. Parenteral formulations are known and are suitable for use in the invention, preferably for i.m. or i.v. administration. The formulations containing therapeutically effective amounts of antibodies or immunotoxins are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients. Lyophilized compositions are reconstituted with suitable diluents, e.g., water for injection, saline, 0.3% glycine and the like, at a level of about from 0.01 mg/kg of host body weight to 10 mg/kg where appropriate. Typically, the pharmaceutical compositions containing the antibodies or immunotoxins will be administered in a therapeutically effective dose in a range of from about 0.01 mg/kg to about 5 mg/kg of the treated mammal. A preferred therapeutically effective dose of the pharmaceutical composition containing antibody or immunotoxin will be in a range of from about 0.01 mg/kg to about 0.5 mg/kg body weight of the treated mammal administered over several days to two weeks by daily intravenous infusion, each given over a one hour period, in a sequential patient dose-escalation regimen.

Antibody may be administered systemically by injection i.m., subcutaneously or intraperitoneally or directly into KS lesions. The dose will be dependent upon the properties of the antibody or immunotoxin employed, e.g., its activity and biological half-life, the concentration of antibody in the formulation, the site and rate of dosage, the clinical tolerance of the patient involved, the disease afflicting the patient and the like as is well within the skill of the physician.

The antibody of the present invention may be administered in solution. The pH of the solution should be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. The antibody or derivatives thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and the like. Buffer concentrations should be in the range of 1 to 100 mM. The solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An effective amount of a stabilizing agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine may also be included and may be added to a solution containing antibody or immunotoxin or to the composition from which the solution is prepared.

Systemic administration of antibody is made daily, generally by intramuscular injection, although intravascular infusion is acceptable. Administration may also be intranasal or by other nonparenteral routes. Antibody or immunotoxin may also be administered via microspheres, liposomes or other microparticulate delivery systems placed in certain tissues including blood.

In therapeutic applications, the dosages of compounds used in accordance with the invention vary depending on the class of compound and the condition being treated. The age, weight, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy are among the factors affecting the selected dosage. For example, the dosage of an immunoglobulin can range from about 0.1 milligram per kilogram of body weight per day to about 10 mg/kg per day for polyclonal antibodies and about 5% to about 20% of that amount for monoclonal antibodies. In such a case, the immunoglobulin can be administered once daily as an intravenous infusion. Preferably, the dosage is repeated daily until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose should be sufficient to treat or ameliorate symptoms or signs of KS without producing unacceptable toxicity to the patient.

An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. The dosing range varies with the compound used, the route of administration and the potency of the particular compound.

VI. Vaccines and Prophylaxis for KS

This invention provides a method of vaccinating a subject against Kaposi's sarcoma, comprising administering to the subject an effective amount of the peptide or polypeptide encoded by the isolated DNA molecule, and a suitable acceptable carrier, thereby vaccinating the subject. In one embodiment naked DNA is administering to the subject in an effective amount to vaccinate a subject against Kaposi's sarcoma.

This invention provides a method of immunizing a subject against a disease caused by the DNA herpesvirus associated with Kaposi's sarcoma which comprises administering to the subject an effective immunizing dose of the isolated herpesvirus vaccine.

A. Vaccines

The invention also provides substances suitable for use as vaccines for the prevention of KS and methods for administering them. The vaccines are directed against the human herpesvirus of the invention, and most preferably comprise antigen obtained from the KS-associated human herpesvirus.

Vaccines can be made recombinantly. Typically, a vaccine will include from about 1 to about 50 micrograms of antigen or antigenic protein or peptide. More preferably, the amount of protein is from about 15 to about 45 micrograms. Typically, the vaccine is formulated so that a dose includes about 0.5 milliliters. The vaccine may be administered by any route known in the art. Preferably, the route is parenteral. More preferably, it is subcutaneous or intramuscular.

There are a number of strategies for amplifying an antigen's effectiveness, particularly as related to the art of vaccines. For example, cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049 which is incorporated by reference herein. More conventionally, an antigen can be conjugated to a suitable carrier, usually a protein molecule. This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule. Additionally, the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.

For parenteral administration, such as subcutaneous injection, examples of suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction. Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.

The conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, "A recent review of protein modification techniques," Bioconjugate Chem. 1:2-12 (1990).

Vaccines against a number of the Herpesviruses have been successfully developed. Vaccines against Varicella-Zoster Virus using a live attenuated Oka strain is effective in preventing herpes zoster in the elderly, and in preventing chickenpox in both immunocompromised and normal children (Hardy, I., et al. [30]; Hardy, I. et al. [31]; Levin, M. J. et al. [54]; Gershon, A. A. [26]. Vaccines against Herpes simplex Types 1 and 2 are also commercially available with some success in protection against primary disease, but have been less successful in preventing the establishment of latent infection in sensory ganglia (Roizman, B. [78]; Skinner, G. R. et al. [87]).

Vaccines against the human herpesvirus can be made by isolating extracellular viral particles from infected cell cultures, inactivating the virus with formaldehyde followed by ultracentrifugation to concentrate the viral particles and remove the formaldehyde, and immunizing individuals with 2 or 3 doses containing 1×10⁹ virus particles (Skinner, G. R. et al. [86]). Alternatively, envelope glycoproteins can be expressed in E. coli or transfected into stable mammalian cell lines, the proteins can be purified and used for vaccination (Lasky, L. A. [53]). MHC--binding peptides from cells infected with the human herpesvirus can be identified for vaccine candidates per the methodology of [61], supra.

The antigen may be combined or mixed with various solutions and other compounds as is known in the art. For example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunodiluting agents. Examples of such adjuvants or agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionibacteriurn acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan). Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Only aluminum is approved for human use.

The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al₂ O₃ basis). On a per-dose basis, the amount of the antigen can range from about 0.1 μg to about 100 μg protein per patient. A preferable range is from about 1 μg to about 50 μg per dose. A more preferred range is about 15 μg to about 45 μg. A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 45 μg of antigen in admixture with 0.5% aluminum hydroxide. After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4° C., or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.

The vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (e.g., subcutaneous or intramuscular) injection. Intramuscular administration is preferred. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time. It is preferred that the dose be given to a human patient within the first 8 months of life. The antigen of the invention can be combined with appropriate doses of compounds including influenza antigens, such as influenza type A antigens. Also, the antigen could be a component of a recombinant vaccine which could be adaptable for oral administration.

Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines. A pharmaceutically effective amount of the antigen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans. Other vaccines may be prepared according to methods well-known to those skilled in the art.

Those of skill will readily recognize that it is only necessary to expose a mammal to appropriate epitopes in order to elicit effective immunoprotection. The epitopes are typically segments of amino acids which are a small portion of the whole protein. Using recombinant genetics, it is routine to alter a natural protein's primary structure to create derivatives embracing epitopes that are identical to or substantially the same as (immunologically equivalent to) the naturally occurring epitopes. Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions of the amino acid sequence for the viral proteins from the human herpesvirus. For example, it is known in the protein art that certain amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein. The human herpesvirus proteins have significant tertiary structure and the epitopes are usually conformational. Thus, modifications should generally preserve conformation to produce a protective immune response.

B. Antibody Prophylaxis

Therapeutic, intravenous, polyclonal or nonoclonal antibodies can been used as a mode of passive immunotherapy of herpesviral diseases including perinatal varicella and CMV. Immune globulin from persons previously infected with the human herpesvirus and bearing a suitably high titer of antibodies against the virus can be given in combination with antiviral agents (e.g. ganciclovir), or in combination with other modes of immunotherapy that are currently being evaluated for the treatment of KS, which are targeted to modulating the immune response (i.e. treatment with copolymer-1, antiidiotypic monoclonal antibodies, T cell "vaccination"). Antibodies to human herpesvirus can be administered to the patient as described herein. Antibodies specific for an epitope expressed on cells infected with the human herpesvirus are preferred and can be obtained as described above.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

C. Monitoring therapeutic efficacy

This invention provides a method for monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma, which comprises determining in a first sample from a subject with Kaposi's sarcoma the presence of the isolated DNA molecule, administering to the subject a therapeutic amount of an agent such that the agent is contacted to the cell in a sample, determining after a suitable period of time the amount of the isolated DNA molecule in the second sample from the treated subject, and comparing the amount of isolated DNA molecule determined in the first sample with the amount determined in the second sample, a difference indicating the effectiveness of the agent, thereby monitoring the therapeutic efficacy of treatment for Kaposi's sarcoma. As defined herein "amount" is viral load or copy number. Methods of determining viral load or copy number are known to those skilled in the art.

VII. Screening Assays For Pharmaceutical Agents of Interest in Alleviating the Symptoms of KS.

Since an agent involved in the causation or progression of KS has been identified and described here, assays directed to identifying potential pharmaceutical agents that inhibit the biological activity of the agent are possible. KS drug screening assays which determine whether or not a drug has activity against the virus described herein are contemplated in this invention. Such assays comprise incubating a compound to be evaluated for use in KS treatment with cells which express the KS associated human herpesvirus proteins or peptides and determining therefrom the effect of the compound on the activity of such agent. In vitro assays in which the virus is maintained in suitable cell culture are preferred, though in vivo animal models would also be effective.

Compounds with activity against the agent of interest or peptides from such agent can be screened in in vitro as well as in vivo assay systems. In vitro assays include infecting peripheral blood leukocytes or susceptible T cell lines such as MT-4 with the agent of interest in the presence of varying concentrations of compounds targeted against viral replication, including nucleoside analogs, chain terminators, antisense oligonucleotides and random polypeptides (Asada, H. et al. [7]; Kikuta et al. [48] both incorporated by reference herein). Infected cultures and their supernatants can be assayed for the total amount of virus including the presence of the viral genome by quantitative PCR, by dot blot assays, or by using immunologic methods. For example, a culture of susceptible cells could be infected with the human herpesvirus in the presence of various concentrations of drug, fixed on slides after a period of days, and examined for viral antigen by indirect immunofluorescence with monoclonal antibodies to viral peptides ([48], supra. Alternatively, chemically adhered MT-4 cell monolayers can be used for an infectious agent assay using indirect immunofluorescent antibody staining to search for focus reduction (Higashi, K. et al. [36], incorporated by reference herein).

As an alternative to whole cell in vitro assays, purified enzymes isolated from the human herpesvirus can be used as targets for rational drug design to determine the effect of the potential drug on enzyme activity, such as thymidine phosphotransferase or DNA polymerase. The genes for these two enzymes are provided herein. A measure of enzyme activity indicates effect on the agent itself.

Drug screens using herpes viral products are known and have been previously described in EP 0514830 (herpes proteases) and WO 94/04920 (U_(L) 13 gene product).

This invention provides an assay for screening anti-KS chemotherapeutics. Infected cells can be incubated in the presence of a chemical agent that is a potential chemotherapeutic against KS (e.g. acyclo-guanosine). The level of virus in the cells is then determined after several days by IFA for antigens or Southern blotting for viral genome or Northern blotting for MRNA and compared to control cells. This assay can quickly screen large numbers of chemical compounds that may be useful against KS.

Further, this invention provides an assay system that is employed to identify drugs or other molecules capable of binding to the DNA molecule or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating transcriptional activity. Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.

This invention is further illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter

EXPERIMENTAL DETAILS SECTION I Experiment 1 Representational difference analysis (RDA) to identify and characterize unique DNA sequences in KS tissue

To search for foreign DNA sequences belonging to an infectious agent in AIDS-KS, representational difference analysis (RDA) was employed to identify and characterize unique DNA sequences in KS tissue that are either absent or present in low copy number in non-diseased tissue obtained from the same patient [58]. This method can detect adenovirus genome added in single copy to human DNA but has not been used to identify previously uncultured infectious agents. RDA is performed by making simplified "representations" of genomes from diseased and normal tissues from the same individual through PCR amplification of short restriction fragments. The DNA representation from the diseased tissue is then ligated to a priming sequence and hybridized to an excess of unligated, normal tissue DNA representation. Only unique sequences found in the diseased tissue have priming sequences on both DNA strands and are preferentially amplified during subsequent rounds of PCR amplification. This process can be repeated using different ligated priming sequences to enrich the sample for unique DNA sequences that are only found in the tissue of interest.

DNA (10 μg) extracted from both the KS lesion and unaffected tissue were separately digested to completion with Bam HI (20 units/μg) at 37° C. for 2 hours and 2 μg of digestion fragments were ligated to NBam12 and NBam24 priming sequences [primer sequences described in 58]. Thirty cycles of PCR amplification were performed to amplify "representations" of both genomes. After construction of the genomic representations, KS tester amplicons between 150 and 1500 bp were isolated from an agarose gel and NBam priming sequences were removed by digestion with Bam HI. To search for unique DNA sequences not found in non-KS driver DNA, a second set of priming sequences (JBam12 and JBam24) was ligated onto only the KS tester DNA amplicons (FIG. 1, lane 1). 0.2 μg of ligated KS lesion amplicons were hybridized to 20 μg of unligated, normal tissue representational amplicons. An aliquot of the hybridization product was then subjected to 10 cycles of PCR amplification using JBam24, followed by mung bean nuclease digestion. An aliquot of the mung bean-treated difference product was then subjected to 15 more cycles of PCR with the JBam24 primer (FIG. 1, lane 2). Amplification products were redigested with Bam HI and 200 ng of the digested product was ligated to RBam12 and RBam24 primer sets for a second round of hybridization and PCR amplification (FIG. 1, lane 3). This enrichment procedure was repeated a third time using the JBam primer set (FIG. 1, lane 4). Both the original driver and the tester DNA samples (Table 2, Patient A) were subsequently found to contain the AIDS-KS specific sequences KS330Bam and KS631Bam (previously identified as KS627Bam) indicating that RDA can be successfully employed when the target sequences are present in unequal copy number in both tissues.

The initial round of DNA amplification-hybridization from KS and normal tissue resulted in a diffuse banding pattern (FIG. 1, lane 2), but four bands at approximately 380, 450, 540 and 680 bp were identifiable after the second amplification-hybridization (FIG. 1, lane 3). These bands became discrete after a third round of amplification-hybridization (FIG. 1, lane 4). Control RDA, performed by hybridizing DNA extracted from AIDS-KS tissue against itself, produced a single band at approximately 540 bp (FIG. 1, lane 5). The four KS-associated bands (designated KS330Bam, KS390Bam, KS480Bam, KS627Bam after digestion of the two flanking 28 bp ligated priming sequences with Bam HI) were gel purified and cloned by insertion into the pCRII vector. PCR products were cloned in the pCRII vector using the TA cloning system (Invitrogen Corporation, San Diego, Calif.).

Experiment 2 Determination of the specificity of AIDS-KS unique sequences.

To determine the specificity of these sequences for AIDS-KS, random-primed ³² P-labeled inserts were hybridized to Southern blots of DNA extracted from cryopreserved tissues obtained from patients with and without AIDS. All AIDS-KS specimens were examined microscopically for morphologic confirmation of KS and immunohistochemically for Factor VIII, Ulex europaeus and CD34 antigen expression. One of the AIDS-KS specimens was apparently mislabeled since KS tissue was not detected on microscopic examination but was included in the KS specimen group for purposes of statistical analysis. Control tissues used for comparison to the KS lesions included 56 lymphomas from patients with and without AIDS, 19 hyperplastic lymph nodes from patients with and without AIDS, 5 vascular tumors from nonAIDS patients and 13 tissues infected with opportunistic infections that commonly occur in AIDS patients. Control DNA was also extracted from a consecutive series of 49 surgical biopsy specimens from patients without AIDS. Additional clinical and demographic information on the specimens was not collected to preserve patient confidentiality.

The tissues, listed in Table 1, were collected from diagnostic biopsies and autopsies between 1983 and 1993 and stored at -70° C. Each tissue sample was from a different patient, except as noted in Table 1. Most of the 27 KS specimens were from lymph nodes dissected under surgical conditions which diminishes possible contamination with normal skin flora. All specimens were digested with Bam HI prior to hybridization. KS390Bam and KS480Bam hybridized nonspecifically to both KS and non-KS tissues and were not further characterized. 20 of 27 (74%) AIDS-KS DNAs hybridized with variable intensity to both KS330Bam and KS627Bam, and one additional KS specimen hybridized only to KS627Bam by Southern blotting (FIG. 2 and Table 1). In contrast to AIDS-KS lesions, only 6 of 39 (15%) non-KS tissues from patients with AIDS hybridized to the KS330Bam and KS627Bam inserts (Table 1).

Specific hybridization did not occur with lymphoma or lymph node DNA from 36 persons without AIDS or with control DNA from 49 tissue biopsy specimens obtained from a consecutive series of patients. DNA extracted from several vascular tumors, including a hemangiopericytoma, two angiosarcomas and a lymphangioma, were also negative by Southern blot hybridization. DNA extracted from tissues with opportunistic infections common to AIDS patients, including 7 acid-fast bacillus (undetermined species), 1 cytomegalovirus, 1 cat-scratch bacillus, 2 cryptococcus and 1 toxoplasmosis infected tissues, were negative by Southern blot hybridization to KS330Bam and KS627Bam (Table 1).

                  TABLE 1                                                          ______________________________________                                         Southern blot hybridization for KS330Bam and                                     KS627Bam and PCR amplification for KS330.sub.234                               in human tissues from individual patients                                                          KS330Bam KS627Bam                                            Southern Southern KS330.sub.234                                                hybridization hybridization PCR                                              Tissue n n (%) n (%) positive                                                ______________________________________                                         AIDS-KS   27*     20 (74)    21 (78) 25 (93)                                     AIDS 27†  3 (11)  3 (11)  3 (11)                                        lymphomas                                                                      AIDS 12  3 (25)  3 (25)  3 (25)                                                lymph nodes                                                                    Non-AIDS 29 0 (0) 0 (0) 0 (0)                                                  Lymphomas                                                                      Non-AIDS 7 0 (0) 0 (0) 0 (0)                                                   lymph nodes                                                                    Vascular 4§ 0 (0) 0 (0) 0 (0)                                             tumors                                                                         Opportunistic 13Π 0 (0) 0 (0) 0 (0)                                         infections                                                                     Consecutive 49¶** 0 (0) 0 (0) 0 (0)                                  surgical biopsies                                                            ______________________________________                                    

Legend to Table 1:

* Includes one AIDS-KS specimen unamplifiable for p53 exon 6 and one tissue which on microscopic examination did not have any detectable KS tissue present. Both of these samples were negative by Southern blot hybridization to KS330Bam and KS627Bam and by PCR amplification for the KS330₂₃₄ amplicon.

† Includes 7 small non-cleaved cell lymphomas, 20 diffuse large cell and immunoblastic lymphomas. Three of the lymphomas with immunoblastic morphology were positive for KS330Bam and KS627Bam.

‡ Includes 13 anaplastic large cell lymphomas, 4 diffuse large cell lymphomas, 4 small lymphocytic lymphomas/chronic lymphocytic leukemias, 3 hairy cell leukemias, 2 monocytoid B-cell lymphomas, 1 follicular small cleaved cell lymphoma, 1 Burkitt's lymphoma, 1 plasmacytoma.

§ Includes 2 angiosarcomas, 1 hemangiopericytoma and 1 lymphangioma.

Π Includes 2 cryptococcus, 1 toxoplasmosis, 1 cat-scratch bacillus, 1 cytomegalovirus, 1 Epstein-Barr virus, and 7 acid-fast bacillus infected tissues. In addition, pure cultures of Mycobacterium avium-complex were negative by Southern hybridization and PCR, and pure cultures of Mycoplasma penetrans were negative by PCR.

¶ Tissues included skin, appendix, kidney, prostate, hernia sac, lung, fibrous tissue, gallbladder, colon, foreskin, thyroid, small bowel, adenoid, vein, axillary tissue, lipoma, heart, mouth, hemorrhoid, pseudoaneurysm and fistula track. Tissues were collected from a consecutive series of biopsies on patients without AIDS but with unknown HIV serostatus.

** Apparent nonspecific hybridization at approximately 20 Kb occurred in 4 consecutive surgical biopsy DNA samples: one colon and one hernia sac DNA sample hybridized to KS330Bam alone, another hernia sac DNA sample hybridized to KS627Bam alone and one appendix DNA sample hybridized to both KS330Bam and KS627Bam. These samples did not hybridize in the 330-630 bp range expected for these sequences and were PCR negative for KS330₂₃₄.

In addition, DNA from Epstein-Barr virus-infected peripheral blood lymphocytes and pure cultures of Mycobacterium avium-complex were also negative by Southern hybridization. Overall, 20 of 27 (74%) AIDS-KS specimens hybridized to KS330Bam and 21 of 27 (78%) AIDS-KS specimens hybridized to KS627Bam, compared to only 6 of 142 (4%) non-KS human DNA control specimens (χ² =85.02, p<10-7 and χ² =92.4, p<10-7 respectively).

The sequence copy number in the AIDS-KS tissues was estimated by simultaneous hybridization with KS330Bam and a 440 bp probe for the constant region of the T cell receptor β gene [76]. Samples in lanes 5 and 6 of FIGS. 2A-2B showed similar intensities for the two probes indicating an average copy number of approximately two KS330Bam sequences per cell, while remaining tissues had weaker hybridization signals for the KS330Bam probe.

Experiment 3 Characterization of KS330Bam and KS627Bam

To further characterize KS330Bam and KS627Bam, six clones for each insert were sequenced. The Sequenase version 2.0 (United States Biochemical, Cleveland, Ohio) system was used and sequencing was performed according to manufacturer's instructions. Nucleotides sequences were confirmed with an Applied Biosystems 373A Sequencer in the DNA Sequencing Facilities at Columbia University.

KS330Bam is a 330 bp sequence with 51% G:C content (FIG. 3B) and KS627Bam is a 627 bp sequence with a 63% G:C content (FIG. 3C). KS330Bam has 54% nucleotide identity to the BDLF1 open reading frame (ORF) of Epstein-Barr virus (EBV). Further analysis revealed that both KS330Bam and KS627Bam code for amino acid sequences with homology to polypeptides of viral origin. SwissProt and PIR protein databases were searched for homologous ORF using BLASTX [3].

KS330Bam is 51% identical by amino acid homology to a portion of the ORF26 open reading frame encoding the capsid protein VP23 (NCBI g.i. 60348, bp 46024-46935) of herpesvirus saimiri [2], a gammaherpesvirus which causes fulminant lymphoma in New world monkeys. This fragment also has a 39% identical amino acid sequence to the theoretical protein encoded by the homologous open reading frame BDLF1 in EBV (NCBI g.i. 59140, bp 132403-133307) [9]. The amino acid sequence encoded by KS627Bam is homologous with weaker identity (31%) to the tegument protein, gp140 (ORF 29, NCBI g.i. 60396, bp108782-112681) of herpesvirus saimiri.

Sequence data from KS330Bam was used to construct PCR primers to amplify a 234 bp fragment designated KS330₂₃₄ (FIG. 3B). The conditions for PCR analyses were as follows: 94° C. for 2 min (1 cycle); 94° C. for 1 min, 58° C. for 1 min, 72° C. for 1 min (35 cycles) ; 72° C. extension for 5 min (1 cycle). Each PCR reaction used 0.1 μg of genomic DNA, 50 pmoles of each primer, 1 unit of Taq polymerase, 100 μM of each deoxynucleotide triphosphate, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), and 0.1% Triton-X-100 in a final volume of 25 μl. Amplifications were carried out in a Perkin-Elmer 480 Thermocycler with 1-s ramp times between steps.

Although Southern blot hybridization detected the KS330Bam sequence in only 20 of 27 KS tissues, 25 of the 27 tissues were positive by PCR amplification for KS330₂₃₄ (FIGS. 4A-4B) demonstrating that KS330Bam is present in some KS lesions at levels below the threshold for detection by Southern blot hybridization. All KS330₂₃₄ PCR products hybridized to a ³² P end-labelled 25 bp internal oligomer, confirming the specificity of the PCR (FIG. 4B). Of the two AIDS-KS specimens negative for KS330₂₃₄, both specimens appeared to be negative for technical reasons: one had no microscopically detectable KS tissue in the frozen sample (FIGS. 4A-4B, lane 3), and the other (FIGS. 4A-4B, lane 15) was negative in the control PCR amplification for the p53 gene indicating either DNA degradation or the presence of PCR inhibitors in the sample. PCR amplification of the p53 tumor suppressor gene was used as a control for DNA quality. Sequences of p53 primers from PE-5, 5'-ACAGGGCTGGTTGCCCAGGGT-3' (SEQ ID No: 44); and P6-3. 5'-AGTTGCAAACCAGACCTCAG-3' (SEQ ID NO: 45) [25].

Except for the 6 control samples from AIDS patients that were also positive by Southern blot hybridization, none of the other 136 control specimens were positive by PCR for KS330₂₃₄. All of these specimens were amplifiable for the p53 gene, indicating that inadequate PCR amplification was not the reason for lack of detection of KS330₂₃₄ in the control tissues. Samples containing DNA from two candidate KS agents, EBV and Mycoplasma penetrans (ATCC Accession No. 55252), a pathogen commonly found in the genital tract of patients with AIDS-KS [59] were also negative for amplification of KS330₂₃₄. In addition, several KS specimens were tested using commercial PCR primers (Stratagene, La Jolla, Calif.) specific for mycoplasmata and primers specific for the EBNA-2, EBNA-3C and EBER regions of EBV and were negative [57].

Overall, DNA from 25 (93%) of 27 AIDS-KS tissues were positive by PCR compared with DNA from 6 (4%) of 142 control tissues, including 6 (15%) of 39 non-KS lymph nodes and lymphomas from AIDS patients (χ² =38.2, p<10⁻⁶), 0 of 36 lymph nodes and lymphomas from nonAIDS patients (χ² =55.2, p<10⁻⁷) and 0 of 49 consecutive biopsy specimens (χ² =67.7, p<10⁻⁷). Thus, KS330₂₃₄ was found in all 25 amplifiable tissues with microscopically detectable AIDS-KS, but rarely occurred in non-KS tissues, including tissues from AIDS patients.

Of the six control tissues from AIDS patients that were positive by both PCR and Southern hybridization, two patients had KS elsewhere, two did not develop KS and complete clinical histories for the remaining two patients were unobtainable. Three of the six positive non-KS tissues were lymph nodes with follicular hyperplasia taken from patients with AIDS. Given the high prevalence of KS among patients with AIDS, it is possible that undetected microscopic foci of KS were present in these lymph nodes. The other three positive tissue specimens were B cell immunoblastic lymphomas from AIDS patients. It is possible that the putative KS agent is also a cofactor for a subset of AIDS-associated lymphomas [16, 17, 80].

To determine whether KS330Bam and KS627Bam are portions of a larger genome and to determine the proximity of the two sequences to each other, samples of KS DNA were digested with Pvu II restriction enzymes. Digested genomic DNA from three AIDS-KS samples were hybridized to KS330Bam and KS627Bam by Southern blotting (FIG. 5). These sequences hybridized to various sized fragments of the digested KS DNA indicating that both sequences are fragments of larger genomes. Differences in the KS330Bam hybridization pattern to Pvu II digests of the three AIDS-KS specimens indicate that polymorphisms may occur in the larger genome. Individual fragments from the digests failed to simultaneously hybridize with both KS330Bam and KS627Bam, demonstrating that these two Bam HI restriction fragments are not adjacent to one another.

If KS330Bam and KS627Bam are heritable polymorphic DNA markers for KS, these sequences should be uniformly detected at non-KS tissue sites in patients with AIDS-KS. Alternatively, if KS330Bam and KS627Bam are sequences specific for an exogenous infectious agent, it is likely that some tissues are uninfected and lack detectable KS330Bam and KS627Bam sequences. DNA extracted from multiple uninvolved tissues from three patients with AIDS-KS were hybridized to ³² P-labelled KS330Bam and KS627Bam probes as well as analyzed by PCR using the KS330₂₃₄ primers (Table 2). While KS lesion DNA samples were positive for both bands, unaffected tissues were frequently negative for these sequences. KS lesions from patients A, B and C, and uninvolved skin and muscle from patient A were positive for KS330Bam and KS627Bam, but muscle and brain tissue from patient B and muscle, brain, colon, heart and hilar lymph node tissues from patient C were negative for these sequences. Uninvolved stomach lining adjacent to the KS lesion in patient C was positive by PCR, but negative by Southern blotting which suggests the presence of the sequences in this tissue at levels below the detection threshold for Southern blotting.

                  TABLE 2                                                          ______________________________________                                         Differential detection of KS330Bam, KS627Bam                                     and KS330.sub.234 sequences in KS-involved and                                 non-involved tissues from three patients with AIDS-KS                                   KS330Bam    KS627Bam  KS330.sub.234                                 ______________________________________                                         Patient A                                                                        KS, skin +  + +                                                                nl skin + + +                                                                  nl muscle + + +                                                                Patient B                                                                      KS, skin + + +                                                                 nl muscle - - -                                                                nl brain - - -                                                                 Patient C                                                                      KS, stomach + + +                                                              nl stomach - - +                                                               adjacent to KS                                                                 nl muscle - - -                                                                nl brain - - -                                                                 nl colon - - -                                                                 nl heart - - -                                                                 nl hilar lymph - - -                                                           nodes                                                                        ______________________________________                                    

Experiment 4 Subcloning and sequencing of KSHV

KS330Bam and KS627Bam are genomic fragments of a novel 5 infectious agent associated with AIDS-KS. A genomic library from a KS lesion was made and a phage clone with a 20 kb insert containing the KS330Bam sequence was identified. The 20 kb clone digested with PvuII (which cuts in the middle of the KS330Bam sequence) 10 produced 1.1 kb and 3 kb fragments that hybridized to KS330Bam. The 1.1 kb subcloned insert and ˜900 bp from the 3 kb subcloned insert resulting in 9404 bp of contiguous sequence was entirely sequenced. This sequence contains partial and complete open reading frames homologous to regions in gamma herpesviruses.

The KS330Bam sequence is an internal portion of an 918 bp ORF with 55-56% nucleotide identity to the ORF26 and BDLF1 genes of HSVSA and EBV respectively. The EBV and HSVSA translated amino acid sequences for these ORFs demonstrate extensive homology with the amino acid sequence encoded by the KS-associated 918 bp ORF (FIG. 6). In HSVSA, the VP23 protein is a late structural protein involved in capsid construction. Reverse transcriptase (RT)-P(R of mRNA from a KS lesion is positive for transcribed KS330Bam mRNA and that indicates that this ORF is transcribed in KS lesions. Additional evidence for homology between the KS agent and herpesviruses comes from a comparison of the genomic organization of other potential ORFs on the 9404 bp sequence (FIG. 3A) The 5' terminus of the sequence is composed nucleotides having 66-67% nucleotide identity and 68-71% amino acid identity to corresponding regions of the major capsid protein (MCP) ORFs for both EBV and HSVSA. This putative MCP ORF of the KS agent lies immediately 5' to the BDLF1/ORF26 homolog which is a conserved orientation among herpesvirus subfamilies for these two genes. At the 3' end of this sequence, the reading frame has strong amino acid and nucleotide homology to HSVSA ORF 27. Thus, KS-associated DNA sequences at four loci in two separate regions with homologies to gamma herpesviral genomes have been identified.

In addition to fragments obtained from Pvu II digest of the 21 Kb phage insert described above, fragments obtained from a BamHI/NotI digest were also subcloned into pBluescript (Stratagene, La Jolla, Calif.). The termini of these subcloned fragments were sequenced and were also found to be homologous to nucleic acid sequence EBV and HSVSA genes. These homologs have been used to develop a preliminary map of subcloned fragments (FIG. 9). Thus, sequencing has revealed that the KS agent maintains co-linear homology to gamma herpesviruses over the length of the 21 Kb phage insert.

Experiment 5 Determination of the phylogeny of KSHV

Regions flanking KS330Bam were sequenced and characterized by directional walking. This was performed by the following strategy: 1) KS genomic libraries were made and screened using the KS330Bam fragment as a hybridization probe, 2) DNA inserts from phage clones positive for the KS330Bam probe were isolated and digested with suitable restriction enzyme(s), 3) the digested fragments were subcloned into pBluescript (Stratagene, La Jolla, Calif.), and 4) the subclones were sequenced. Using this strategy, the major capsid protein (MCP) ORF homolog was the first important gene locus identified. Using sequenced unique 3' and 5' end-fragments from positive phage clones as probes, and following the strategy above a KS genomic library are screened by standard methods for additional contiguous sequences.

For sequencing purposes, restriction fragments are subcloned into phagemid pBluescript KS+, pBluescript KS-, pBS+, or pBS- (Stratagene) or into plasmid pUC18 or pUC19. Recombinant DNA was purified through CsCl density gradients or by anion-exchange chromatography (Qiagen).

Nucleotide sequenced by standard screening methods of cloned fragments of KSHV were done by direct sequencing of double- stranded DNA using oligonucleotide primers synthesized commercially to "walk" along the fragments by the dideoxy-nucleotide chain termination method. Junctions between clones are confirmed by sequencing overlapping clones.

Targeted homologous genes in regions flanking KS330Bam include, but are not limited to: Il-10 homolog, thymidine kinase (TK), g85 g35, gH, capsid proteins and MCP. TK is an early protein of the herpesviruses functionally linked to DNA replication an a target enzyme for anti-herpesviral nucleosides. TK phosphorylates acyclic nucleosides such as acyclovir which in turn inhibit viral DNA polymerase chain extension. Determining the sequence of this gene will aid in the prediction of chemotherapeutic agents useful against KSHV. TK is encoded by the EBV BXLF1 ORF located ˜9700 bp rightward of BDLF1 and by the HSVSA ORF 21 ˜9200 bp rightward of the ORF 26. A subcloned fragment of KS5 was identified with strong homology to the EBV and HSVSA TK open reading frames.

g85 is a late glycoprotein involved in membrane fusion homologous to gH in HSV1. In EBV, this protein is encoded by BLXF2 ORF located ˜7600 bp rightward of BDLF1, and in HSVSA it is encoded by ORF 22 located ˜7100 bp rightward of ORF26.

g35 is a late EBV glycoprotein found in virion and plasma membrane. It is encoded by BDLF3 ORF which is 1300 bp leftward of BDLF1 in EBV. There is no BDLF3 homolog in HSVSA. A subcloned fragment has already been identified with strong homology to the EBV gp35 open reading frame.

Major capsid protein (MCP) is a conserved 150 KDa protein which is the major component of herpesvirus capsid. Antibodies are generated against the MCP during natural infection with most herpesviruses. The terminal 1026 bp of this major capsid gene homolog in KSHV have been sequenced.

Targeted homologous genes/loci in regions flanking KS627Bam include, but are not limited to: terminal reiterated repeats, LMPI, EBERs and Ori P. Terminal reiterated sequences are present in all herpesviruses. In EBV, tandomly reiterated 0.5 Kb long terminal repeats flank the ends of the linear genome and become joined in the circular form. The terminal repeat region is immediately adjacent to BNRF1 in EBV and ORF 75 in HSVSA. Since the number of terminal repeats varies between viral strains, identification of terminal repeat regions may allow typing and clonality studies of KSHV in KS legions. Sequencing through the terminal repeat region may determine whether this virus is integrated into human genome in KS.

LMPI is an latent protein important in the transforming effects of EBV in Burkitt's lymphoma. This gene is encoded by the EBV BNRF1 ORF located ˜2000 bp rightward of tegument protein ORF BNRF1 in the circularized genome. There is no LMP1 homolog in HSVSA.

EBERs are the most abundant RNA in latently EBV infected cells and Ori-P is the origin of replication for latent EBV genome. This region is located between ˜4000-9000 bp leftward of the BNRF1 ORF in EBV; there are no corresponding regions in HSVSA.

The data indicates that the KS agent is a new human herpesvirus related to gamma herpesviruses EBV and HSVSA. The results are not due to contamination or to incidental co-infection with a known herpesvirus since the sequences are distinct from all sequenced herpesviral genomes (including EBV, CMV, HHV6 and HSVSA) and are associated specifically with KS in three separate comparative studies. Furthermore, POR testing of KS DNA with primers specific for EBV-1 and EBV-2 failed to demonstrate these viral genomes in these tissues. Al though KSHV is homologous to EBV regions, the sequence does not match any other known sequence and thus provides evidence for a new viral genome, related to but distinct from known members of the herpesvirus family.

Experiment 6 Serological studies

Indirect immunofluorescence assay (IFA)

Virus-containing cells are coated to a microscope slide. The slides are treated with organic fixatives, dried and then incubated with patient sera. Antibodies in the sera bind to the cells, and then excess nonspecific antibodies are washed off. An antihuman immunoglobulin linked to a fluorochrome, such as fluorescein, is then incubated with the slides, and then excess fluorescent immunoglobulin is washed off. The slides are then examined under a microscope and if the cells fluoresce, then this indicates that the sera contains antibodies directed against the antigens present in the cells, such as the virus.

An indirect immunofluorescence assay (IFA) was performed on the Body Cavity-Based Lymphoma cell line (BCBL-1), which is a naturally transformed EBV infected (nonproducing) B cell line, using 4 KS patient sera and 4 control sera (from AIDS patients without KS). Initially, both sets of sera showed similar levels of antibody binding. To remove nonspecific antibodies directed against EBV and lymphocyte antigens, sera at 1:25 dilution were pre-adsorbed using 3×10⁶ 1% paraformaldehyde-fixed Raji cells per ml of sera. BCBL1 cells were fixed with ethanol/acetone, incubated with dilutions of patient sera, washed and incubated with fluorescein-conjugated goat anti-human IgG. Indirect immunofluorescent staining was determined.

Table 3 shows that unabsorbed case and control sera have similar end-point dilution indirect immunofluorescence assay (IFA) titers against the BCBL1 cell line. After Raji adsorption, case sera have four-fold higher IFA titers against BCBL1 cells than control sera. Results indicated that pre-adsorption against paraformaldehyde-fixed Raji cells reduces fluorescent antibody binding in control sera but do not eliminate antibody binding to KS case sera. These results indicate that subjects with KS have specific antibodies directed against the KS agent that can be detected in serological assays such as IFA, Western blot and Enzyme immunoassays (Table 3).

                  TABLE 3                                                          ______________________________________                                         Indirect immunofluorescence end-point titers                                     for KS case and non-KS control sera against                                    the BCBL-1 cell line                                                             Sera No.  Status*  Pre-adsorption                                                                          Post-adsorption**                              ______________________________________                                         1         KS       ≧1:400                                                                             ≧1:400                                      2 KS  1:100  1:100                                                             3 KS  1:200  1:100                                                             4 KS ≧1:400  1:200                                                      5 Control ≧1:400 1:50                                                   6 Control 1:50 1:50                                                            7 Control  1:100 1:50                                                          8 Control  1:200 1:50                                                        ______________________________________                                          Legend Table 3:                                                                *KS = autopsyconfirmed male, AIDS patient                                      Control = autopsyconfirmed female, AIDS patient, no KS                         **Adsorbed against RAJI cells treated with 1% paraformaldehyde           

Immunoblotting ("Western blot")

Virus-containing cells or purified virus (or a portion of the virus, such as a fusion protein) is electrophoresed on a polyacrylamide gel to separate the protein antigens by molecular weight. The proteins are blotted onto a nitrocellulose or nylon membrane, then the membrane is incubated in patient sera. Antibodies directed against specific antigens are developed by incubating with a anti-human immunoglobulin attached to a reporter enzyme, such as a peroxidase. After developing the membrane, each antigen reacting against antibodies in patient sera shows up as a band on the membrane at the corresponding molecular weight region.

Enzyme immunoassay ("EIA or ELISA")

Virus-containing cells or purified virus (or a portion of the virus, such as a fusion protein) is coated to the bottom of a 96-well plate by various means (generally incubating in alkaline carbonate buffer). The plates are washed, then the wells are incubated with patient sera. Antibodies in the sera directed against specific antigens stick on the plate. The wells are washed again to remove nonspecific antibody, then they are incubated with a antihuman immunoglobulin attached to a reporter enzyme, such as a peroxidase. The plate is washed again to remove nonspecific antibody and then developed. Wells containing antigen that is specifically recognized by antibodies in the patients sera change color and can be detected by an ELISA plate reader (a spectrophotomer).

All three of these methods can be made more specific by pre-incubating patient sera with uninfected cells to adsorb out cross-reacting antibodies against the cells or against other viruses that may be present in the cell line, such as EBV. Cross-reacting antibodies can potentially give a falsely positive test result (i.e. the patient is actually not infected with the virus but has a positive test result because of cross-reacting antibodies directed against cell antigens in the preparation). The importance of the infection experiments with Raji is that if Raji cells, or another well-defined cell line, can be infected, then the patient's sera can be pre-adsorbed against the uninfected parental cell line and then tested in one of the assays. The only antibodies left in the sera after pre-adsorption that bind to antigens in the preparation should be directed against the virus.

Experiment 7

BCBL 1, from lymphomatous tissues belonging to a rare infiltrating, anaplastic body cavity lymphoma occurring in AIDS patients has been placed in continuous cell culture and shown to be continuously infected with the KS agent. This cell line is also naturally infected with Epstein-Barr Virus (EBV). The BCBL cell line was used as an antigen substrate to detect specific KS antibodies in persons infected with the putative virus by Western-blotting. Three lymphoid B cell lines were used as controls. These included the EBV genome positive cell line P3H3, the EBV genome defective cell line Raji and the EBV genome negative cell line Bjab.

Cells from late-log phase culture were washed 3 time with PBS by centrifugation at 500 g for 10 min. and suspended in sample buffer containing 50 mM Tris-HCl pH 6.8, 2% SDS (w/v), 15% glycerol (v/v), 5% β-mercaptoethanol (v/v) and 0.001% bromophenol (w/v) with protease inhibitor, 100 μM phenylmethylsulfonyl fluoride (PMSF). The sample was boiled at 100° C. for 5 min and centrifuged at 14,000 g for 10 min. The proteins in the supernatant was then fractionated by sodium, dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions with a separation gel of 15% and a stacking gel of 5% (3). Prestained protein standards were included: myosin, 200 kDa; β-galactosidase, 118 kDA; BSA, 78 kDa; ovalbumin, 47.1 kDa; carbonic anhydrase, 31.4 kDa; soybean trypsin inhibitor, 25.5 kDa, lysozyme, 18.8 kDa and aprotinin, 8.3 kDa (Bio-Rad). Immunoblotting experiments were performed according to the method of Towbin et al. (4). Briefly, the proteins were electrophorectically transferred to Hybon-C extra membranes (Pharmacia) at 24 V for 70 min. The membranes were then dried at 37° C. for 30 min, saturated with 5% skim milk in Tris-buffered saline, pH 7.4 (TBS) containing 50 mM Tris-HCl and 200 mM NaCl, at room temperature for 1 h. The membranes were subsequently incubated with human sera at dilution 1:200 in 1% skim milk overnight at room temperature, washed 3 times with a solution containing TBS, 0.2% Triton X-100 and 0.05% skim milk and then 2 times with TBS. The membranes were then incubated for 2 h at room temperature with alkaline phosphatase conjugated goat anti-mouse IgG+IgM+IgA (Sigma) diluted at 1:5000 in 1% skim milk. After repeating the washing,the membranes were stained with nitroblue tetranolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL).

Two bands of approximately 226 kDa and 234 kDa were identified to be specifically present on the Wester-blot of BCBL cell lysate in 5 sera from AIDS gay man patients infected with KS. These 2 bands were absent from the lysates of P3H3, Raji and Bjab cell lysates. 5 sera from AIDS gay man patients without KS and 2 sera from AIDS woman patients without KS as well as 1 sera from nasopharyncel carcinoma patient were not able to detect these 2 bands in BCBL 1, P3H3, Raji and Bjab cell lysates. In a blinded experiment, using the 226 kDa and 234 kDa markers, 15 out of 16 sera from KS patients were correctly identified. In total, the 226 kDa and 234 kDa markers were detected in 20 out of 21 sera from KS patients.

The antigen is enriched in the nuclei fraction of BCBL1. Enriched antigen with low background can be obtained by preparing nucleic from BCBC as the starting antigen preparation using standard, widely available protocols. For example, 500-750 ml of BCBL at 5×10⁵ cells/ml can be pelleted at low speed. The pellet is placed in 10 mM NaCl, 10 mM Tris pH 7.8, 1.5 mM MgCl₂ (equi volume)+1.0% NP-40 on ice for 20 min to lyse cells. The lysate is then spun at 1500 rpm for 10 min. to pellet nucleic. The pellet is used as the starting fraction for the antigen preparation for the Western blot. This will reduce cross- reactive cytoplasmic antigens.

Experiment 8 Transmission studies

Co-infection experiments

BCBL1 cells were co-cultivated with Raji cell lines separated by a 0.45μ tissue filter insert. Approximately, 1-2×10⁶ BCBL1 and 2×10⁶ Raji cells were co-cultivated for 2-20 days in supplemented RPMI alone, in 10 μg/ml 5'-bromodeoxyuridine (BUdR) and 0.6 μg/ml 5'-flourodeoxyuridine or 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). After 2,8,12 or 20 days co-cultivation, Raji cells were removed, washed and placed in supplemented RPMI 1640 media. A Raji culture co-cultivated with BCBL1 in 20 ng/ml TPA for 2 days survived and has been kept in continuous suspension culture for >10 weeks. This cell line, designated RCC1 (Raji Co-Culture, No. 1) remains PCR positive for the KS330₂₃₄ sequence after multiple passages. This cell line is identical to its parental Raji cell line by flow cytometry using EMA, B1, B4 and BerH2 lymphocyte-flow cytometry (approximately 2%). RCC1 periodically undergo rapid cytolysis suggestive of lytic reproduction of the agent. Thus, RCC1 is a Raji cell line newly infected with KSHV.

The results indicate the presence of a new human virus, specifically a herpesvirus in KS lesions. The high degree of association between this agent and AIDS-KS (>90%), and the low prevalence of the agent in non-KS tissues from immunocompromised AIDS patients, indicates that this agent has a causal role in AIDS-KS [47, 68].

Experiment 10 Isolation of KSHV

Crude virus preparations are made from either the supernatant or low speed pelleted cell fraction of BCBL1 cultures. Approximately 650 ml or more of log phase cells should be used (>5×10⁶ cells/ml).

For bonding whole virion from supernatant, the cell free supernatant is spun at 10,000 rpm in a GSA rotor for 10 min to remove debris. PEG-8000 is added to 7%, dissolved and placed on ice for >2.5 hours. The PEG-supernatant is then spun at 10,000×g for 30 min. supernatant is poured off and the pellet is dried and scraped together from the centrifuge bottles. The pellet is then resuspended in a small volume (1-2 ml) of virus buffer (VB, 0.1 M NaCl, 0.01 M Tris, pH 7.5). This procedure will precipitate both naked genome and whole virion. The virion are then isolated by centrifugation at 25,000 rpm in a 10-50% sucrose gradient made with VB. One ml fractions of the gradient are then obtained by standard techniques (e.g. using a fractionator) and each fraction is then tested by dot blotting using specific hybridizing primer sequences to determine the gradient fraction containing the purified virus (preparation of the fraction maybe needed in order to detect the presence of the virus, such as standard DNA extraction).

To obtain the episomal DNA from the virus, the pellet of cells is washed and pelleted in PBS, then lysed using hypotonic shock and/or repeated cycles of freezing and thawing in a small volume (<3 ml). Nuclei and other cytoplasmic debris are removed by centrifugation at 10,000 g for 10 min, filtration through a 0.45 m filter and then repeat centrifugation at 10,000 g for 10 min. This crude preparation contains viral genome and soluble cell components. The genome preparation can then be gently chloroform-phenol extracted to remove associated proteins or can be placed in neutral DNA buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA, pH 7.2-7.6) with 2% sodium dodecylsulfate (SDS) and 1% sarcosyl. The genome is then banded by centrifugation through 10-30% sucrose gradient in neutral DNA buffer containing 0.15% sarcosyl at 20,000 rpm in a SW 27.1 rotor for 12 hours (for 40,000 rpm for 2-3 hours in an SW41 rotor). The band is detected as described above.

An example of the method for isolating KSHV genome from KSHV infect ed cell cultures (97 and 98). Approximately 800 ml of BCBL1 cells are pelleted, washed with saline, and pelleted by low speed centrifugation. The cell pellet is lysed with an equal volume of RSB (10 mM NaCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 7.8) with 1% NP-40 on ice for 10 minutes. The lysate is centrifuged at 900×g for 10 minutes to pellet nuclei. This step is repeated. To the supernatant is added 0.4% sodium dodecylsulfate and EDTA to a final concentration of 10 mM. The supernatant is loaded on a 10-30% sucrose gradient in 1.0 M NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.5. The gradients are centrifuged at 20,000 rpm on a SW 27.1 rotor for 12 hours. In FIG. 11, 0.5 ml aliquots of the gradient have been fractionated (fractions 1-62) with the 30% gradient fraction being at fraction No. 1 and the 10%. gradient fraction being at fraction No. 62. Each fraction has been dot hybridized to a nitrocellulose membrane and then a ³² P-labeled KSHV DNA fragment, KS631Bam has been hybridized to the membrane using standard techniques. FIG. 11 shows that the major solubilized fraction of the KSHV genome bands (i.e. is isolated) in fractions 42 through 48 of the gradient with a high concentration of the genome being present in fraction 44. A second band of solubilized KSHV DNA occurs in fractions 26 through 32.

EXPERIMENT 11 Purification of KSHV

DNA is extracted using standard techniques from the RCC-1 or RCC-1_(2F5) cell line [27, 49, 66]. The DNA is tested for the presence of the KSHV by Southern blotting and PCR using the specific probes as described hereinafter. Fresh lymphoma tissue containing viable infected cells is simultaneously filtered to form a single cell suspension by standard techniques [49, 66]. The cells are separated by standard Ficoll-Plaque centrifugation and lymphocyte layer is removed. The lymphocytes are then placed at >1×10⁶ cells/ml into standard lymphocyte tissue culture medium, such as RMP 1640 supplemented with 10% fetal calf serum. Immortalized lymphocytes containing the KSHV virus are indefinitely grown in the culture media while nonimmortilized cells die during course of prolonged cultivation.

Further, the virus may be propagated in a new cell line by removing media supernatant containing the virus from a continuously infected cell line at a concentration of >1×10⁶ cells/ml. The media is centrifuged at 2000×g for 10 minutes and filtered through a 0.45μ filter to remove cells. The media is applied in a 1:1 volume with cells growing at >1×10⁶ cells/ml for 48 hours. The cells are washed and pelleted and placed in fresh culture medium, and tested after 14 days of growth.

The herpesvirus may be isolated from the cell DNA in the following manner. An infected cell line, which can be lysed using standard methods such as hyposmotic shocking and Dounce homogenization, is first pelleted at 2000×g for 10 minutes, the supernatant is removed and centrifuged again at 10,000×g for 15 minutes to remove nuclei and organelles. The supernatant is filtered through a 0.45μ filter and centrifuged again at 100,000×g for 1 hour to pellet the virus. The virus can then be washed and centrifuged again at 100,000×g for 1 hour.

REFERENCES

1. Ablashi, D. V., et al. Virology 184:545-552.

2. Albrecht, J. C., et al. (1992) J. Virol. 66:5047.

3. Altshul, S. F., et al. (1990) J. Molec. Biol. 215:403.

4. Analytical Biochemistry (1984) 238:267-284.

5. Andrei, et al. (1992) Eur. J. Clin. Microbiol. Infect. Dis. 11(2):143-51.

6. Archibald, C. P., et al. (1992) Epidemiol. 3:203.

7. Asada, H., et al (1989) J. Clin. Microbiol. 27(10):2204.

8. Ausubel, F., et al. (1987) Current Protocols in Molecular Biology, New York.

9. Baer, R. J., et al. (1984) Nature 310:207.

10. Bagasra, et al. (1992) J. New England Journal of Medicine 326(21):1385-1391.

11. Balzarini, et al. (1990) Mol. Pharm. 37,402-7.

12. Basic and Clinical Immunology 7th Edition D. Stites and A. Terr ed.

13. Beral, V., et al. (1990) Lancet 335:123.

14. Beral, V., et al. (1991) Brit. Med. J. 302:624.

15. Beral, V., et al. (1992) Lancet 339:632.

16. Bendsoe, N., et al. (1990) Eur. J. Cancer 26:699.

17. Biggar, R. J., et al. (1994) Am. J. Epidemiol. 139:362.

18. Bovenzi, P., et al. (1993) Lancet 341:1288.

19. Beaucage and Carruthers (1981) Tetrahedron Lett. 22:1859-1862.

20. Braitman, et al. (1991) Antimicrob. Agents and Chemotherapy 35(7):1464-8.

21. Burns and Sanford, (1990) J. Infect. Dis. 162(3):634-7.

22. De Clercq, (1993) Antimicrobial Chemotherapy 32, Suppl. A, 121-132.

23. Drew, W. L., et al. (1982) Lancet ii:125.

24. Falk, et al. (1991) Nature 351:290.

25. Gaidano, G., et al. (1991) Proc. Natl. Acad. Sci. USA 88:5413.

26. Gershon, A. A., (1992) J. Inf. Des. 166(Suppl):563.

27. Glick, J. L., (1980) Fundamentals of Human Lymphoid Culture, Marcel Dokker, New York.

28. Gorbach, S. L., et al. (1992) Infectious Disease Ch.35:289, W. B. Saunders, Philadelphia, Pa.

29. Greenspan, et al. (1990) J. Acquir. Immune Defic. Syndr. 3 (6):571.

30. Hardy, I., et al. (1990) Inf. Dis. Clin. N. Amer. 4(1):159.

31. Hardy, I., et al. (1991) New Engl. J. Med. 325 (22):1545.

31A. Harel-Bellan, A., et al. (1988) Exp. Med. 168:2309-2318

32. Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York.

33. Haverkos, H. W., et al. (1985) Sexually Transm. Dis. 12:203.

34. Helene, C. and Toulme, J. (1990) Biochim. Biophys. Acta. 1049:99-125.

35. Heniford, et al. (1993) Nucleic Acids Research 21(14):3159-3166.

36. Higashi, K., et al. (1989) J. Clin. Micro. 27(10):2204.

37. Holmberg, S. D., et al. (1990) Cancer Detection and Prevention 14:331.

38. Holliday, J., and Williams, M. V., (1992) Antimicrob. Agents Chemother. 36(9):1935.

39. Hoogenboom, H. R., et al. (1991) Nuc. Acids Res. 19:4133.

40. Hunt, et al. (1991) Eur. J. Immunol. 21:2963-2970.

41. Hybridization of Nucleic Acids Immobilized on Solid Supports Meinkoth, J. and Wahl, G.

42. Hybridization with Nucleic Acid Probes pp. 495-524, (1993) Elsevier, Amsterdam.

43. Ickes, et al. (1994) Antiviral Research 23, Seventh International Conf. on Antiviral Research, Abstract No. 122, Supp. 1.

44. Jahan, N., et al. (1989) AIDS Research and Human Retroviruses 5:225.

45. Jardetzkey, et al. (1991) Nature 353:326.

46. Johnston, G. S., et al. (1990) Cancer Detection and Prevention 14:337.

47. Jung, J. U., et al. (1991) Proc. Natl. Acad. Sci. USA 88:7051.

48. Kikuta, et al. (1989) Lancet Oct. 7:861.

49. Knowles, D. M., et al. (1989) Blood 73:792-798.

50. Kohler and Milstein, (1976) Eur. J. Immunol. 6:511-519.

51. Kucera, et al. (1993) AIDS Res. Human Retroviruses 9:307-314.

52. Laboratory Techniques in Biochemistry and Molecular Biology (1978) North Holland Publishing Company, New York.

53. Lasky, L. A., (1990) J. Med. Virol. 31(1):59.

54. Levin, M. J., et al. (1992) J. Inf. Dis. 166(2):253.

55. Lifson, A. R., et al. (1990) Am. J. Epidemiol. 131:221.

56. Lin, et al. (1991) Antimicrob Agents Chemother 35(11):2440-3.

57. Lin, J. C., et al. (1993) Blood 81:3372.

58. Lisitsyn, N., et al. (1993) Science 259:946.

59. Lo, S -C., et al. (1992) Internat. J. Systematic Bacteriol. 42:357.

60. Marks, J. D., et al. (1991) J. Mol Biol. 222:581-597.

61. Marloes, et al. (1991) Eur. J. Immunol. 21:2963-2970.

62. Matteucci, et al. (1981) Am. Chem. Soc. 103:3185.

63. Maxam, A. M. and Gilbert, W. Methods in Enzymology (1980) Grossman, L. and Moldave, D., eds., Academic Press, New York, 65:499-560.

64. McCafferty, J., et al. (1990) Nature 348:552.

65. Means and Feeney, (1990) Bioconjugate Chem. A recent reveiw of protein modification techniques, 1:2-12.

66. Metcalf, D. (1984) Clonal Culture of Hematopoeitic Cells:Techniques and Applications, Elvier, New York.

67. Methods in Enzymology Vol. 152, (1987) Berger, S. and Kimmel, A. ed., Academic Press, New York

68. Miller, G., Virology (1990) B. N. Fields, D. M. Knipe eds., Raven Press, New York, 2:1921.

69. Needham-VanDevanter, D. R., et al., (1984) Nucelic Acids Res. 12:6159-6168.

70. Needleman and Wunsch, (1970) J. Mol. Biol. 48:443.

71. Neuvo, et al. (1993) American Journal of Surgical Pathology 17(7), 683-690.

72. Nucleic Acid Hybridization: A Practical Approach (1985) Ed. Hames, B. D. and Higgins, S. J., IRL Press.

73. Oren and Soble, (1991) Clinical Infectious Diseases 14:741-6.

74. PCR Protocols: A Guide to Methods and Applications. (1990) Innis, M., Gelfand D., Sninsky, J. and White, T., eds., Academic Press, San Diego.

75. Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. (USA) 85:2444.

75A. Pearson, J. D., and Regnier, F. E., (1983) J. Chrom. 255:137-14976.

76. Pellici, P. G., et al. (1985) J. Exp. Med. 162:1015.

77. Peterman, T. A., et al. (1991) Cancer Surveys Imperial Cancer Research Fund, London, 10:23-37.

78. Roizman, B. (1991) Rev. Inf. Disease 13 Suppl. 11:S892.

79. Rotzschke and Falk, (1991) Immunol. Today 12:447.

80. Safai, B., et al. (1980) Cancer 45:147:2.

81. Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3.

82. Saunders, et al. (1990) J. Acquir. Immune Defic. Syndr. 3 (6):571.

83. Schecter, M. T., et al. (1991) Am. J. Epidemiol. 134:485.

84. Scopes, R., (1982) Protein Purification: Principles and Practice Springer-Verlag, New York.

85. Siddiqui, A., et al. (1983) Proc. Natl. Acad. Sci. USA 80:4861.

86. Skinner, G. R., et al. (1991) Comp. Immuno. Microbiol. Inf. Dis. 14(2):13.

87. Skinner, G. R., et al. (1992) Med. Microbiol. Immunol. 180(6):305. Smith and Waterman (1981) Adv. Appl. Math. 2:482.

88. Snoeck, et al. (1992) Eur. J. Clin. Micro. Infect. Dis. 11(12):1144-55.

89. Stals, et al. (1993) Antimicrobial Agents Chemother. 37(2):218-23.

90. van den Berg, F. et al. (1989) J. Clin. Pathol. 42:128.

91. Vogel, J., et al. (1988) Nature 335:606.

92. Wang, R. H. -Y., et al. (1993) Clin. Infect. Dis. 17:724.

93. Wickstrom, E. L., et al. (1988) PNAS (USA) 85:1028-1032.

94. Winkelmann, et al. (1988) Drug Res. 38, 1545-48.

95. Winkler, et al. (1990) Antiviral Research 14:61-74.

96. Yamandaka, et al. (1991) Mol. Pharmacol. 40(3):446.

97. Pellicer, A. et al. (1978) Cell 14:133-141.

98. Gibson, W. and Roizmann B. (1972) J. Virol. 10:1044-52.

EXPERIMENTAL DETAILS SECTION II

Sequencing Studies:

A lambda phage (KS5) from a KS lesion genomic library identified by positive hybridization with KS330Bam was digested with BamHI and Not I (Boehringer-Mannheim, Indianapolis Ind.); five fragments were gel isolated and subcloned into Bluescript II KS (Stratagene, La Jolla Calif.). The entire sequence was determined by bidirectional sequencing at a seven fold average redundancy by primer walking and nested deletions.

DNA sequence data were compiled and aligned using ALIGN (IBI-Kodak, Rochester N.Y.) and analyzed using the Wisconsin Sequence Analysis Package Version 8-UNIX (Genetics Computer Group, Madison Wis.) and the GRAIL Sequence Analysis, Gene Assembly and Sequence Comparison System v. 1.2 (Informatics Group, Oak Ridge Tenn.). Protein site motifs were identified using Motif (Genetics Computer Group, Madison Wis.).

Sources of Herpesvirus Gene Sequence Comparisons:

Complete genomic sequences of three gammaherpesviruses were available: Epstein-Barr virus (EBV), a herpesvirus of humans [4]; herpesvirus saimiri (HVS), a herpesvirus of the New World monkey Saimiri sciureus [1]; and equine herpesvirus 2 (EHV2 [49]). Additional thymidine kinase gene sequences were obtained for alcelaphine herpesvirus 1 (AHV1 [22]) and bovine herpesvirus 4 (BHV4 [31]). Sequences for the major capsid protein genes of human herpesvirus 6B and human herpesvirus 7 (HHV7) were from Mukai et al. [34]. The sources of all other sequences used are listed previously in McGeoch and Cook [31] and McGeoch et al. [32].

Phylogenetic Inference:

Predicted amino acid sequences used for tree construction were based on previous experience with herpesviral phylogenetic analyses [31]. Alignments of homologous sets of amino acid sequences were made with the AMPS [5] and Pileup [16] programs. Regions of alignments that showed extreme divergence with marked length heterogeneity, typically terminal sections, were excised. Generally, positions in alignments that contained inserted gaps in one or more sequences were removed before use for tree construction. Phylogenetic inference programs were from the Phylip set, version 3.5c [14] and from the GCG set [16]. Trees were built with the maximum parsimony (MP), neighbor joining (NJ) methods. For the NJ method, which utilizes estimates of pairwise distances between sequences, distances were estimated as mean numbers of substitution events per site with Protdist using the PAM 250 substitution probability matrix of Schwartz & Dayhoff [46]. Bootstrap analysis [15] was carried out for MP and NJ trees, with 100 sub-replicates of each alignment, and consensus trees obtained with the program Consense. In addition the program Protml was used to infer trees by the maximum likelihood (ML) method. Protml was obtained form J. Adachi, Department of Statistical Science, The Graduate University for Advanced Study, Tokyo 106, Japan. Because of computational constraints, Protml was used only with the 4-species CS1 alignment.

Clamped Homogeneous Electric Field (CHEF) Gel Electrophoresis:

Agarose plugs were prepared by resuspending BCBL-1 cells in 1% LMP agarose (Biorad, Hercules Calif.) and 0.9% NaCl at 42° C. to a final concentration of 2.5×10⁷ cells/ml. Solidified agarose plugs were transferred into lysis buffer (0.5M EDTA pH 8.0, 1% sarcosyl, proteinase K at 1 mg/ml final concentration) and incubated for 24 hours. Approximately 10⁷ BCBL-1 cells were loaded in each lane. Gels were run at a gradient of 6.0 V/cm with a run time of 28 h 28 min. on a CHEF Mapper XA pulsed field gel electrophoresis apparatus (Biorad, Hercules Calif.), Southern blotted and hybridized to KS627Bam, KS330Bam and an EBV terminal repeat sequence [40].

TPA Induction of Genome Replication:

Late Log phase BCBL-1 cells (5×10 cells per ml) were incubated with varying amounts of 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma Chemical Co., St. Louis Mo.) for 48 h, cells were then harvested and washed with phosphate-buffered saline (PBS) and DNA was isolated by chloroform-phenol extraction. DNA concentrations were determined by UV absorbance; 5 μg of whole cell DNA was quantitatively dot blot hybridized in triplicate (Manifold I, Schleicher and Schuell, Keene NH). KS631Bam, EBV terminal repeat and beta-actin sequences were random-primer labeled with ³² P [13]. Specific hybridization was quantitated on a Molecular Dynamics PhosphorImager 425E.

Cell Cultures and Transmission Studies:

Cells were maintained at 5×10⁵ cells per ml in RPMI 1640 with 20% fetal calf serum (FCS, Gibco-BRL, Gaithersburg Md.) and periodically examined for continued KSHV infection by PCR and dot hybridization. The T cell line Molt-3 (a gift from Dr. Jodi Black, Centers for Disease Control and Prevention), Raji cells (American Type Culture Collection, Rockville Md.) and RCC-1 cells were cultured in RPMI 1640 with 10% FCS. Owl monkey kidney cells (American Type Culture Collection, Rockville Md.) were cultured in MEM with 10% FCS and 1% nonessential amino acids (Gibco-BRL, Gaithersburg Md.).

To produce the RCC-1 cell line, 2×10⁶ Raji cells were cultivated with 1.4×10⁶ BCBL-1 cells in the presence of 20 ng/ml TPA for 2 days in chambers separated by Falcon 0.45 μg filter tissue culture inserts to prevent contamination of Raji with BCBL-1. Demonstration that RCC-1 was not contaminated with BCBL-1 was obtained by PCR typing of HLA-DR alleles [27] (Raji and RCC-1: DRβ1*0310, DRβ3*02, BCBL-1: DRβ104,*07, Drβ4*01) and confirmed by flow cytometry to determine the presence (Raji, RCC1) or absence (BCBL-1) of EMA membrane antigen. Clonal sublines of RCC-1 were obtained by dilution in 96 well plates to 0.1 cells/well in RPMI 1640, 20% FCS and 30% T-STIM culture supplement (Collaborative Biomedical Products, Bedford Mass.). Subcultures were examined to ensure that each was derived from a single cluster of growing cells.

In situ hybridization was performed with a previously described 25 bp oligomer located in ORF26 which was 5' labeled with fluorescein (Operon, Alameda Calif.) and hybridized to cytospin preparations of BCBL-1, RCC-1 and Raji cells using the methods of Lungu et al. [29]. Slides were both directly visualized by UV microscopy and by incubating slides with anti-fluorescein-alkaline phosphatase (AP)-conjugated antibody (Boehringer-Mannheim, Indianapolis Ind.), allowing immunohistochemical detection of bound probe. Positive control hybridization was performed using a 26 bp TET-labeled EBV DNA polymerase gene oligomer (Applied Biosystems, Alameda Calif.) which was visualized by UV microscopy only and negative control hybridization was performed using a 25 bp 5' fluorescein-labeled HSV1 α47 gene oligomer (Operon, Alameda Calif.) which was visualized in a similar manner as the KSHV ORF26 probe. All nuclei of BCBL-1, RCC-1 and Raji appropriately stained with the EBV hybridization probe whereas no specific staining of the cells occurred after hybridization with the HSV1 probe.

The remaining suspension cell lines used in transmission experiments were pelleted, and resuspended in 5 ml of 0.22 or 0.45μ filtered BCBL-1 tissue culture supernatant for 16 h. BCBL-1 supernatants were either from unstimulated cultures or from cultures stimulated with 20 ng/ml TPA. No difference in transmission to recipient cell lines was noted using various filtration or stimulation conditions. Fetal cord blood lymphocytes (FCBL) were obtained from heparinized fresh post-partum umbilical cord blood after separation on Ficoll-Paque (Pharmacia LKB, Uppsala Sweden) gradients and cultured in RPMI 1640 with 10% fetal calf serum. Adherent recipient cells were washed with sterile Hank's Buffered Salt Solution (HBSS, Gibco-BRL, Gaithersburg Md.) and overlaid with 5 ml of BCBL-1 media supernatant. After incubation with BCBL-1 media supernatant, cells were washed three times with sterile HBSS, and suspended in fresh media. Cells were subsequently rewashed three times every other day for six days and grown for at least two weeks prior to DNA extraction and testing. PCR to detect KSHV infection was performed using nested and unnested primers from ORF 26 and ORF 25 as previously described [10, 35].

Indirect Immunofluorescence Assay:

AIDS-KS sera were obtained from ongoing cohort studies (provided by Drs. Scott Holmberg, Thomas Spira and Harold Jaffe, Centers for Disease Control, and Prevention, and Isaac Weisfuse, New York City Department of Health). Sera from AIDS-KS patients were drawn between 1 and 31 months after initial KS diagnosis, sera from intravenous drug user and homosexual/bisexual controls were drawn after non-KS AIDS diagnosis, and sera from HIV-infected hemophiliac controls were drawn at various times after HIV infection. Immunofluorescence assays were performed using an equal volume mixture of goat anti-human IgG-FITC conjugate (Molecular Probes, Eugene Oreg.) and goat anti-human IgM-FITC conjugate (Sigma Chemical Co., St. Louis Mo.) diluted 1:100 and serial dilutions of patient sera. End-point titers were read blindly and specific immunoglobulin binding was assessed by the presence or absence of a specular fluorescence pattern in the nuclei of the plated cells. To adsorb cross-reacting antibodies, 20 μl serum diluted 1:10 in phosphate-buffer saline (PBS), pH 7.4, were adsorbed with 1-3×10⁷ paraformaldehyde-fixed P3H3 cells for 4-10 h at 25° C. and removed by low speed centrifugation. P3H3 were induced prior to fixation with 20 ng/ml TPA for 48 h, fixed with 1% paraformaldehyde in PBS for 2 h at 4° C., and washed three times in PBS prior to adsorption.

RESULTS

Sequence Analysis of a 20.7 kb KSHV DNA Sequence:

To demonstrate that KS330Bam and KS631Bam are genomic fragments from a new and previously uncharacterized herpesvirus, a lambda phase clone (KS5) derived from an AIDS-KS genomic DNA library was identified by hybridization to the KS330Bam sequence. The KS5 insert was subcloned after NotI/BamHI digestion into five subfragments and both strands of each fragment were sequenced by primer walking or nested deletion with a 7-fold average redundancy. The KS5 sequence is 20,705 bp in length and has a G+C content of 54.0%. The observed/expected CpG dinucleotide ratio is 0.92 indicating no overall CpG suppression in this region.

Open reading frame (ORF) analysis identified 15 complete ORFs with coding regions ranging from 231 bp to 4128 bp in length, and two incomplete ORFs at the termini of the KS5 clone which were 135 and 552 bp in length (FIG. 12). The coding probability of each ORF was analyzed using GRAIL 2 and CodonPreference which identified 17 regions having excellent to good protein coding probabilities. Each region is within an ORF encoding a homolog to a known herpesvirus gene with the exception of one ORF located at the genome position corresponding to ORF28 in herpesvirus saimiri (HVS). Codon preference values for all of the ORFs were higher across predicted ORFs than in non-coding regions when using a codon table composed of KS5 homologs to the conserved herpesvirus major capsid (MCP), glycoprotein H (gH), thymidine kinase (TK), and the putative DNA packaging protein (ORF29a/ORF29b) genes.

The translated sequence of each ORF was used to search GenBank/EMBL databases with BLASTX and FastA algorithms [2, 38]. All of the putative KS5 ORFs, except one, have sequence and collinear positional homology to ORFs from gamma-2 herpesviruses, especially HVS and equine herpesvirus 2 (EHV2). Because of the high degree of collinearity and amino acid sequence similarity between KSHV and HVS, KSHV ORFs have been named according to their HVS positional homologs (i.e. KSHV ORF25 is named after HVS ORF 25).

The KS5 sequence spans a region which includes three of the seven conserved herpesvirus gene blocks (FIG. 14) [10]. ORFs present in these blocks include genes which encode herpesvirus virion structural proteins and enzymes involved in DNA metabolism and replication. Amino acid identities between KS5 ORFs and HVS ORFs range from 30% to 60%, with the conserved MCP ORF25 and ORF29b genes having the highest percentage amino acid identity to homologs in other gammaherpesviruses. KSHV ORF28, which has no detectable sequence homology to HVS or EBV genes, has positional homology to HVS ORF28 and EBV BDLF3. ORF28 lies at the junction of two gene blocks (FIG. 14); these junctions tend to exhibit greater sequence divergence than intrablock regions among herpesviral genomes [17]. Two ORFs were identified with sequence homology to the putative spliced protein packaging genes of HVS (ORF29a/ORF29b) and herpes simplex virus type 1 (UL15). The KS330Bam sequence is located within KSHV ORF26, whose HSV-1 counterpart, VP23, is a minor virion structural component.

For every KSHV homolog, the HVS amino acid similarity spans the entire gene product, with the exception of ORF21, the TK gene. The KSHV TK homolog contains a proline-rich domain at its amino terminus (nt 20343-19636; aa 1-236) that is not conserved in other herpesvirus TK sequences, while the carboxyl terminus (nt 19637-18601; aa 237-565) is highly similar to the corresponding regions of HVS, EHV2, and bovine herpesvirus 4 (BHV4) TK. A purine binding motif with a glycine-rich region found in herpesviral TK genes, as well as other TK genes, is present in the KSHV TK homolog (GVMGVGKS; aa 260-267).

The KS5 translated amino acid sequences were searched against the PROSITE Dictionary of Protein Sites and Patterns (Dr. Amos Bairoch, University of Geneva, Switzerland) using the computer program Motifs. Four sequence motif matches were identified among KSHV hypothetical protein sequences. These matches included: (i) a cytochrome c family heme-binding motif in ORF33 (CVHCHG; aa 209-214 (SEQ ID NO:11)) and ORF34 (CLLCHI; aa 257-261 (SEQ ID NO:35)), (ii) an immunoglobulin and major histocompatibility complex protein signature in ORF25 (FICQAKH; aa 1024-1030 (SEQ ID NO:3)), (iii) a mitochondrial energy transfer protein motif in ORF26 (PDDITRMRV; aa 260-268 (SEQ ID NO:25)), and (iv) the purine nucleotide binding site identified in ORF21. The purine binding motif is the only motif with obvious functional significance. A cytosine-specific methylase motif present in HVS ORF27 is not present in KSHV ORF27. This motif may play a role in the methylation of episomal DNA in cells persistently infected with HVS [1].

Phylogenetic Analysis of KSHV:

Amino acid sequences translated from the KS5 sequence were aligned with corresponding sequences from other herpesviruses. On the basis of the level of conserved aligned residues and the low incidence of introduced gaps, the amino acid alignments for ORFs 21, 22, 23, 24, 25, 26, 29a, 29b, 31 and 34 were suitable for phylogenetic analyses.

To demonstrate the phylogenetic relationship of KSHV to other herpesviruses, a single-gene comparison was made for ORF25 (MCP) homologs from KS5 and twelve members of Herpesviridae (FIGS. 15A-15B) . The thirteen available MCP amino acid sequences are large (1376 a.a. residues for the KSHV homolog) and alignment required only a low level of gapping; however, the overall similarity between viruses is relatively low [33]. The MCP set gave stable trees with high bootstrap scores and assigned the KSHV homolog to the gamma-2 sublineage (genus Rhadinovirus), containing HVS, EHV2 and BVH4 [20, 33, 43]. KSHV was most closely associated with HVS. Similar results were obtained for single-gene alignments of TK and UL15/ORF29 sets but with lower bootstrap scores so that among gamma-2 herpesvirus members branching orders for EHV2, HVS and KSHV were not resolved.

To determine the relative divergence between KSHV and other gammaherpesviruses, alignments for the nine genes listed above were concatenated to produce a combined gammaherpesvirus gene set (CS1) containing EBV, EHV2, HVS and KSHV amino acid sequences. The total length of CS1 was 4247 residues after removal of positions containing gaps introduced by the alignment process in one or more of the sequences. The CS1 alignment was analyzed by the ML method, giving the tree shown in FIG. 15B and by the MP and NJ methods used with the aligned herpesvirus MCP sequences. All three methods identified KSHV and HVS as sister groups, confirming that KSHV belongs in the gamma-2 sublineage with HVS as its closest known relative. It was previously estimated that divergence of the HVS and EHV2 lineages may have been contemporary with divergence of the primate and ungulate host lineages [33]. The results for the CS1 set suggest that HVS and KSHV represent a lineage of primate herpesviruses and, based on the distance between KSHV and HVS relative to the position of EHV2, divergence between HVS and KSHV lines is ancient.

Genomic Studies of KSHV:

CHEF electrophoresis performed on BCBL-1 cells embedded in agarose plugs demonstrated the presence of a nonintegrated KSHV genome as well as a high molecular weight species (FIGS. 16A-16B) . KS631Bam (FIG. 16A) and KS330Bam specifically hybridized to a single CHEF gel band comigrating with 270 kilobase (kb) linear DNA standards. The majority of hybridizing DNA was present in a diffuse band at the well origin; a low intensity high molecular weight (HMW) band was also present immediately below the origin (FIG. 16A. arrow). The same filter was stripped and probed with an EBV terminal repeat sequence [40] yielding a 150-160 kb band (FIG. 16B) corresponding to linear EBV DNA [24]. The HMW EBV band may correspond to either circular or concatemeric EBV DNA [24].

The phorbol ester TPA induces replication-competent EBV to enter a lytic replication cycle [49]. To determine if TPA induces replication of KSHV and EBV in BCBL-1 cells, these cells were incubated with varying concentrations of TPA for 48 h (FIG. 17). Maximum stimulation of EBV occurred at 20 ng/ml TPA which resulted in an eight-fold increase in hybridizing EBV genome. Only a 1.3-1.4 fold increase in KSHV genome abundance occurred after 20-80 ng/ml TPA incubation for 48 h.

Transmission Studies:

Prior to determining that the agent was likely to be a member of Herpesviridae by sequence analysis, BCBL-1 cells were cultured with Raji cells, a nonlytic EBV transformed B cell line, in chambers separated by a 0.45μ tissue culture filter. Recipient Raji cells generally demonstrated rapid cytolysis suggesting transmission of a cytotoxic component from the BCBL-1 cell line. One Raji line cultured in 10 ng/ml TPA for 2 days, underwent an initial period of cytolysis before recovery and resumption of logarithmic growth. This cell line (RCC-1) is a monoculture derived from Raji uncontaminated by BCBL-1 as determined by PCR amplification of HLA-DR sequences.

RCC-1 has remained positive for the KS330₂₃₃ PCR product for >6 months in continuous culture (approximately 70 passages), but KSHV was not detectable by dot or Southern hybridization at any time. In situ hybridization, however, with a 25 bp KSHV ORF26-derived oligomer was used to demonstrate persistent localization of KSHV DNA to RCC-1 nuclei. As indicated in FIGS. 18A-18C, nuclei of BCBL-1 and RCC-1 (from passage ˜65) cells had detectable hybridization with the ORF26 oligomer, whereas no specific hybridization occurred with parental Raji cells (FIG. 18B). KSHV sequences were detectable in 65% of BCBL-1 and 2.6% of RCC-1 cells under these conditions. In addition, forty-five monoclonal cultures were subcultured by serial dilution from RCC-1 at passage 50, of which eight (18%) clones were PCR positive by KS330₂₃₃. While PCR detection using unnested KS330₂₃₃ primer pairs was lost by passage 15 in each of the clonal cultures, persistent KSHV genome was detected in 5 clones using two more sensitive nonoverlapping nested PCR primer sets [33] suggesting that KSHV genome is lost over time in RCC-1 and its clones.

Low but persistent levels of KS330₂₃₃ PCR positivity were found for one of four Raji, one of four Bjab, two of three Molt-3, one of one owl monkey kidney cell lines and three of eight human fetal cord blood lymphocyte (FCBL) cultures after inoculation with 0.2-0.45μ filtered BCBL-1 supernatants. Among the PCR positive cultures, PCR detectable genome was lost after 2-6 weeks and multiple washings. Five FCBL cultures developed cell clusters characteristic of EBV immortalized lymphocytes and were positive for EBV by PCR using EBER primers [23); three of these cultures were also initially KS330₂₃₃ positive. None of the recipient cell lines had detectable KSHV genome by dot blot hybridization.

Serologic Studies:

Indirect immunofluorescence antibody assays (IFA) were used to assess the presence of specific antibodies against the KSHV- and EBV-infected cell line HBL-6 in the sera from AIDS-KS patients and control patients with HIV infection or AIDS. HBL-6 was substituted for BCBL-1 for reasons of convenience; preliminary studies showed no significant differences in IFA results between HBL-6 and BCBL-1. HBL-6 have diffuse immunofluorescent cell staining with most KS patient and control unabsorbed sera suggesting nonspecific antibody binding (FIGS. 19A-19D). After adsorption with paraformaldehyde-fixed, TPA-induced P3H3 (an EBV producer subline of P3J-HR1, a gift of Dr. George Miller) to remove cross-reacting antibodies against EBV and lymphocyte antigens, patient sera generally showed specular nuclear staining at high titers while this staining pattern was absent from control patient sera (FIGS. 19B and 19D). Staining was localized primarily to the nucleus but weak cytoplasmic staining was also present at low sera dilutions.

With unadsorbed sera, the initial endpoint geometric mean titers (GMT) against HBL-6 cell antigens for the sera from AIDS-KS patients (GMT=1:1153, range: 1:150 to 1:12,150) were higher than for sera from control, non-KS patients (GMT=1:342; range 1:50 to 1:12,150: p=0.04) (FIG. 13). While AIDS-KS patients and HIV-infected gay/bisexual and intravenous drug user control patients had similar endpoint titers to HBL-6 antigens (GMT=1:1265 and GMT=1:1578, respectively), hemophilic AIDS patient titers were lower (GMT=1:104). Both case and control patient groups had elevated IFA titers against the EBV infected cell line P3H3.

The difference in endpoint GMT between case and control titers against HBL-6 antigens increased after adsorption with P3H3. After adsorption, case GMT declined to 1:780 and control GMT declined to 1:81 (p=0.00009). Similar results were obtained by using BCBL-1 instead of HBL-6 cells, by pre-adsorbing with EBV-infected nonproducer Raji cells instead of P3H3 and by using sera from a homosexual male KS patient without HIV infection, in complete remission for KS for 9 months (HBL-6 titer 1:450, P3H3 titer 1:150). Paired sera taken 8-14 months prior to KS onset and after KS onset were available for three KS patients: KS patients 8 and 13 had eight-fold rises and patient 8 had a three-fold fall in P3H3-adsorbed BCBL-1 titers from pre-onset sera to post-KS sera.

DISCUSSION

These studies demonstrate that specific DNA sequences found in KS lesions by representational difference analysis belong to a newly identified human herpesvirus. The current studies define this agent as a human gamma-2 herpesvirus that can be continuously cultured in naturally-transformed, EBV-coinfected lymphocytes from AIDS-related body-cavity based lymphomas.

Sequence analysis of the KS5 lambda phage insert provides clear evidence that the KS330Bam sequence is part of a larger herpesvirus genome. KS5 has a 54.0% G+C content which is considerably higher than the corresponding HVS region (34.3% G+C) . While there is no CpG dinucleotide suppression in the KS5 sequence, the corresponding HVS region has a 0.33 expected:observed CpG dinucleotide ratio [1]. The CpG dinucleotide frequency in herpesviruses varies from global CpG suppression among gammaherpesviruses to local CpG suppression in the betaherpesviruses, which may result from deamination of 5'-methylcytosine residues at CpG sites resulting in TpG substitutions [21]. CpG suppression among herpesviruses [21, 30, 44] has been hypothesized to reflect co-replication of latent genome in actively dividing host cells, but it is unknown whether or not KSHV is primarily maintained by a lytic replication cycle in vivo.

The 20,705 bp KS5 fragment has 17 protein-coding regions, 15 of which are complete ORFs with appropriately located TATA and polyadenylation signals, and two incomplete ORFs located at the phage insert termini. Sixteen of these ORFs correspond by sequence and collinear positional homology to 15 previously identified herpesviral genes including the highly conserved spliced gene. The conserved positional and sequence homology for KSHV genes in this region are consistent with the possibility that the biological behavior of the virus is similar to that of other gammaherpesviruses. For example, identification of a thymidine kinase-like gene on KS5 implies that the agent is potentially susceptible to TK-activated DNA polymerase inhibitors and like other herpesviruses possesses viral genes involved in nucleotide metabolism and DNA replication [41]. The presence of major capsid protein and glycoprotein H gene homologs suggest that replication competent virus would produce a capsid structure similar to other herpesviruses.

Phylogenetic analyses of molecular sequences show that KSHV belongs to the gamma-2 sublineage of the Gammaherpesvirinae subfamily, and is thus the first human gamma-2 herpesvirus identified. Its closest known relative based on available sequence comparisons is HVS, a squirrel monkey gamma-2 herpesvirus that causes fulminant polyclonal T cell lymphoproliferative disorders in some New World monkey species. Data for the gamma-2 sublineage are sparse: only three viruses (KSHV, HVS and EHV2) can at present be placed on the phylogenetic tree with precision (the sublineage also contains murine herpesvirus 68 and BHV4 [33]. Given the limitation in resolution imposed by this thin background, KSHV and HVS appear to represent a lineage of primate gamma-2 viruses. Previously, McGeoch et al. [33] proposed that lines of gamma-2 herpesviruses may have originated by cospeciation with the ancestors of their host species. Extrapolation of this view to KSHV and HVS suggests that these viruses diverged at an ancient time, possibly contemporaneously with the divergence of the Old World and New World primate host lineages. Gammaherpesviruses are distinguished as a subfamily by their lymphotrophism [41] and this grouping is supported by phylogenetic analysis based or sequence data [33]. The biologic behavior of KSHV is consistent with its phylogenetic designation in that KSHV can be found in in vitro lymphocyte cultures and in in vivo samples of lymphocytes [3].

This band appears to be a linear form of the genome because other "high molecular weight" bands are present for both EBV and KSHV in BCBL-1 which may represent circular forms of their genomes. The linear form of the EBV genome, associated with replicating and packaged DNA [41] migrates substantially faster than the closed circular form associated with latent viral replication [24]. While the 270 kb band appears to be a linear form, it is also consistent with a replicating dimer plasmid since the genome size of HVS is approximately 135 kb. The true size of the genome may only be resolved by ongoing mapping and sequencing studies.

Replication deficient EBV mutants are common among EBV strains passaged through prolonged tissue culture [23]. The EBV strain infecting Raji, for example, is an BALF-2 deficient mutant [19]; virus replication is not inducibile with TPA and its genome is maintained only as a latent circular form [23, 33]. The EBV strain coinfecting BCBL-1 does not appear to be replication deficient because TPA induces eight-fold increases in DNA content and has an apparent linear form on CHEF electrophoresis. KSHV replication, however, is only marginally induced by comparable TPA treatment indicating either insensitivity to TPA induction or that the genome has undergone loss of genetic elements required for TPA induction. Additional experiments, however, indicate that KSHV DNA can be pelleted by high speed centrifugation of filtered organelle-free, DNase I-protected BCBL-1 cell extracts, which is consistent with KSHV encapsidation.

Transmission of KSHV DNA from BCBL-1 to a variety of recipient cell lines is possible and KSHV DNA can be maintained at low levels in recipient cells for up to 70 passages. However, detection of virus genome in recipient cell lines by PCR may be due to physical association of KSHV DNA fragments rather than true infection. This appears to be unlikely given evidence for specific nuclear localization of the ORF26 sequence in RCC-1. If transmission of infectious virus from BCBL-1 occurs, it is apparent that the viral genome declines in abundance with subsequent passages of recipient cells. This is consistent with studies of spindle cell lines derived from KS lesions. Spindle cell cultures generally have PCR detectable KSHV genome when first explanted, but rapidly lose viral genome after initial passages and established spindle cell cultures generally do not have detectable KSHV sequences [3].

Infections with the human herpesviruses are generally ubiquitous in that nearly all humans are infected by early adulthood with six of the seven previously identified human herpesviruses [42]. Universal infection with EBV, for example, is the primary reason for the difficulty in clearly establishing a causal role for this virus in EBV-associated human tumors. The serologic studies identified nuclear antigen in BCBL-1 and HBL-6 which is recognized by sera from AIDS-KS patients but generally not by sera from control AIDS patients without KS after removal of EBV-reactive antibodies. These data are consistent with PCR studies of KS and control patient lymphocytes suggesting that KSHV is not ubiquitous among adult humans, but is specifically associated with persons who develop Kaposi's sarcoma. In this respect, it appears to be epidemiologically similar to HSV2 rather than the other known human herpesviruses. An alternative possibility is that elevated IFA titers against BCBL-1 reflect disease status rather than infection with the virus.

REFERENCES

1. Albrecht, J.-C., J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newamn, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess. 1992. Primary structure of the Herpesvirus saimiri genome. J Virol. 66:5047-5058.

2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J Mol Biol. 215:403-410.

3. Ambroziak, J. A., D. J. Blackbourn, B. G. Herndier, R. G. Glogau, J. H. Gullett, A. R. McDonald, E. T. Lennette, and J. A. Levy. 1995. Herpes-like sequences in HIV-infected and uninfected Kaposi's sarcoma patients. Science. 268:582-583.

4. Baer, R., A. T. Bankier, P. L. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 310:207-211.

5. Barton, G. J., and M. J. E. Sternberg. 1987. A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J Mol Biol. 198:327-37.

6. Beral, V., T. A. Peterman, R. L. Berkelman, and H. W. Jaffe. 1990. Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet. 335:123-128.

7. Boshoff, C., D. Whitby, T. Hatziionnou, C. Fisher, J. van der Walt, A. Hatzakis, R. Weiss, and T. Schulz. 1995. Kaposi's sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet. 345:1043-44.

8. Cesarman, E., Y. Chang, P. S. Moore, J. W. Said, and D. M. Knowles. 1995. Kaposi's sarcoma-associated herpesvirus-like DNA sequences are present in AIDS-related body cavity based lymphomas. New England J Med. 332:1186-1191.

9. Chang, Y., E. Cesarman, M. S. Pessin, F. Lee, J. Culpepper, D. M. Knowles, and P. S. Moore. 1994. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 265:1865-69.

10. Chee, M. S., S. B. Bankier, C. M. Bohni, R. C. Brown, T. Horsnell, C. A. Hutchison, T. Kouzarides, J. A. Martignetti, E. Preddie, S. C. Satchwell, P. Tomlinson, K. M. Weston, and B. G. Barrell. 1990. Analysis of the protein coding content of the sequence of cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 154:125-69.

11. Collandre, H., S. Ferris, O. Grau, L. Montagnier, and A. Blanchard. 1995. Kaposi's sarcoma and new herpesvirus. Lancet. 345:1043.

12. Dupin, N., M. Grandadam, V. Calvez, I. Gorin, J. T. Aubin, S. Harvard, F. Lamy, M. Leibowitch, J. M. Huraux, J. P. Escande, and H. Agut. 1995. Herpesvirus-like DNA in patients with Mediterranean Kaposi's sarcoma. Lancet. 345:761-2.

13. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 132:6.

14. Felsenstein, J. 1989. PHYLIP-phylogeny inference package (ver 3.2). Cladistics. 5:164-6.

15. Felsenstein, J. 1988. Phylogenies from molecular sequences: inferences and reliability. Annual Rev Microbiol. 22:521-65.

16. Genetics Computer Group. 1994. Program manual for the GCG package, version 8, Madison, Wis.

17. Gompels, U. A., J. Nicholas, G. Lawrence, M. Jones, B. J. Thomson, M. E. D. Martin, S. Efstathiou, M. Craxton, and H. A. Macaulay. 1995. The DNA sequence of human herpesvirus-6: Structure, coding content and genome evolution. Virology. 209:29-51.

18. Hatfull, G., A. T. Bankier, B. G. Barrell, and P. J. Farrell. 1988. Sequence analysis of Raji Epstein-Barr virus DNA. Virol. 164:334-40.

19. Holmberg, S. D. 1990. Possible cofactors for the development of AIDS-related neoplasms. Cancer Detection and Prevention. 14:331-336.

20. Honess, R. W. 1984. Herpes simplex and `the herpes complex`: diverse observations and a unifying hypothesis. J Gen Virol. 65:2077-2107.

21. Honess, R. W., U. A. Gompels, B. G. Barrell, M. Craxton, K. R. Cameron, R. Staden, Y.-N. Chang, and G. S. Hayward. 1989. Deviations from expected frequencies of CpG dinucleotides in herpesvirus DNAs; may be diagnostic of differences in the states of their latent genomes. J Gen Virol. 70:837-55.

22. Hsu, D., L. M. Shih, and Y. C. Zee. 1990. Nucleotide sequence of a 3.5 nucleotide fragment of malignant catarrhal fever virus strain WC11. Arch Virol. 113:53-60.

23. Kieff, E., and D. Liebowitz. 1990. Epstein-Barr virus and its replication, p. 1889-1920. In B. N. Fields and D. M. Knipe (ed.), Virology, vol. 2. Raven Press, New York.

24. Kolman, J. L., C. J. Kolman, and G. Miller. 1992. Marked variation in the size of genomic plasmids among members of the family of related Epstein-Barr viruses. Proc Natl Acad Sci, USA. 89:7772-7776.

25. Lebbe, C., P. de Cremoux, M. Rybojad, C. Costa da Cunha, P. Morel, and F. Calvo. 1995. Kaposi's sarcoma and new herpesvirus. Lancet. 345:1180.

26. Lin, J. C., S. C. Lin, B. K. De, W. P. Chan, and B. L. Evatt. 1993. Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C and EBER): predominance of type A virus associated with Hodgkin's disease. Blood. 81:3372-81.

27. Liu, Z., S. Yu-Kai, Y.-P. Xi, P. Harris, and N. Suciu-Foca. 1992. T cell recognition of self-human histocompatibility leukocyte antigens (HLA)-DR peptides in the context of syngeneic HLA-DR molecules. J Exp. Med. 175:1663-8.

28. Lomonte, P., M. Bublot, P--P. Pastoret, and E. Thiry. 1992. Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase of other herpesviruses. Arch. Virol. 127:327-337.

29. Martin, M. E. D., J. Nicholas, B. J. Thomson, C. Newman, and R. W. Honess. 1991. Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6. J Virol. 65:5381-5390.

30. McGeoch, D. J., and S. Cook. 1994. Molecular phylogeny of the Alphaherpesvirinae subfamily and a proposed evolutionary timescale. J Mol Biol. 238:9-22.

31. McGeoch, D. J., S. Cook, A. Dolar., F. E. Jamieson, and E. A. R. Telford. 1995. Molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses. J Molec Biol. 247:443-58.

32. Miller, G. 1990. Epstein-Barr virus: Biology, pathogenesis and medical aspects, p. 1921-1957. In B. N. Fields and D. M. Knipe (ed.), Virology, 2nd ed, vol. 2. Raven Press, New York.

33. Moore, P. S., and Y. Chang. 1995. Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. New England J Med. 332:1181-1185.

34. Mukai, T., Y. Isegawa, and K. Yamanishi. 1995. Identification of the major capsid protein gene of human herpesvirus 7. Virus Res. 37:55-62.

35. Oettle, A. G. 1962. Geographic and racial differences in the frequency of Kaposi's sarcoma as evidence of environmental or genetic causes, vol. 18. Symposium on Kaposi's sarcoma: Unio Internationalis Contra Cancrum, Karger, Basel.

36. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence analysis. Proc Natl Acad Sci, USA. 85:2444-8.

37. Peterman, T. A., H. W. Jaffe, A. E. Friedman-Kien, and R. A. Weiss. 1991. The aetiology of Kaposi's sarcoma, p. 23-37, Cancer, HIV, and AIDS, vol. 10. Imperial Cancer Research Fund. London.

38. Raab-Traub, N., and K. Flynn. 1986. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell. 47:883-889.

39. Roizman, B. 1993. The family Herpesviridae, p. 1-9. In B. Roizman and R. J. Whitley and C. Lopez (ed.), The Human Herpeviruses. Raven Press, Ltd., New York.

40. Roizman, B. 1995. New viral footprints in Kaposi's sarcoma. N Engl J Med. 332:1227-1228.

41. Roizman, B., R. C. Desrosiers, B. Fleckenstein, C. Lopez, A. C. Minson, and M. J. Studdert. 1992. The family Herpesviridae: an update. Arch Virol. 123:425-449.

42. Sandford, G. R., K. Ho, and W. H. Burns. 1993. Characterization of the major locus of immediate-early genes of rat cytomegalovirus. J Virol. 67:4093-4103.

43. Schalling, M., M. Ekman, E. E. Kaaya, A. Linde, and P. Bieberfeld. 1995. A role for a new herpesvirus (KSHV) in different forms of Kaposi's sarcoma. Nature Med. 1:707-8.

44. Schwartz, R. M., and M. O. Dayhoff. 1978. Matrices for detecting distant relationships, p. 353-8. In M. O. Dayhoff (ed.), Atlas of protein sequence and structure, vol. 5, supple 3. National Biomedical Research Foundation, Washington.

45. Su, I.-J., Y.-S. Hsu, Y.-C. Chang, and I.-W. Wang. 1995. Herpesvirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet. 345:722-23.

46. Telford, E. A. R., M. S. Watson, H. C. Aird, J. Perry, and A. J. Davison. 1995. The DNA sequence of equine herpesvirus 2. J Molec Biol. 249:520-8.

47. zur Hausen, H., F. J. O'Neill, and U. K. Freese. 1978. Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature. 272:373-375.

EXPERIMENTAL DETAILS SECTION III

KS Patient Enrollment:

Cases and controls were selected from ongoing cohort studies based on the availability of clinical information and appropriate PBMC samples. 21 homosexual or bisexual men with AIDS who developed KS during their participation in prospective cohort studies were identified [14-16]. Fourteen of these patients had paired PBMC samples collected after KS diagnosis (median+4 months) and at least four months prior to KS diagnosis (median-13 months), while the remaining 7 had paired PBMC taken at the study visit immediately prior to KS diagnosis (median-3 months) and at entry into their cohort study (median-51 months prior to KS diagnosis).

Hemophilic and Homosexual/Bisexual Male AIDS Patient Control Enrollment:

Two control groups of AIDS patients were examined: 23 homosexual/bisexual men with AIDS followed until death who did not develop KS ("high risk" control group) from the Multicenter AIDS Cohort Study [16]), and 19 hemophilic men ("low risk" control group) enrolled from joint projects of the National Hemophilia Foundation and the Centers for Disease Control and Prevention. Of the 16 hemophilic controls with available follow-up information, none are known to have developed KS and <2% of hemophilic AIDS patients historically develop KS [2]. For homosexual/bisexual AIDS control patients who did not develop KS, paired PBMC specimens were available at entry into their cohort study (median-35 months prior to AIDS onset) and at the study visit immediately prior to nonKS AIDS diagnosis (median (HBL-6 months prior to AIDS onset).

DNA Extraction and Analyses:

DNA from 10⁶ -10⁷ PBMC in each specimen was extracted and quantitated by spectrophotometry. Samples were prepared in physically isolated laboratories from the laboratory where polymerase chain reaction (PCR) analyses were performed. All samples were tested for amplifiability using primers specific for either the HLA-DQ locus (GH26/GH27) or b-globin [18]. PCR detection of KSHV DNA was performed as previously described [7] with the following nested primer sets: No. 1 outer 5'-AGCACTCGCAGGGCAGTACG-3' (SEQ ID NO:51) 5'-GACTCTTCGCTGATGAACTGG-3'; No. 1 inner 5'-TCCGTGTTGTCTACGTCCAG-3' (SEQ ID NO:52), 5'-AGCCGAAAGGATTCCACCAT-3' (SEQ ID NO:53); No. 2 outer 5'-AGGCAACGTCAGATGTGAC-3' (SEQ ID NO:41), 5'-GAAATTACCCACGAGATCGC-3' (SEQ ID NO:54); No. 2 inner 5'-CATGGGAGTACATTGTCAGGACCTC-3' (SEQ ID NO:42), 5'-GGAATTATCTCGCAGGTTGCC-3' (SEQ ID NO:55); No. 3 outer 5'-GGCGACATTCATCAACCTCAGGG-3' (SEQ ID NO:56), 5'-ATATCATCCTGTGCGTTCACGAC-3' (SEQ ID NO:57); No. 3 inner 5'-CATGGGAGTACATTGTCAGGACCTC-3' (SEQ ID NO:58); 5'-GGAATTATCTCGCAGGTTGCC-3' (SEQ ID NO:56). The outer primer set was amplified for 35 cycles at 94° C. for 30 seconds, 60° C. for 1 minute and 72° C. for 1 minute with a 5 minute final extension cycle at 72° C. One to three ml of the PCR product was added to the inner PCR reaction mixture and amplified for 25 additional cycles with a 5 minute final extension cycle. Primary determination of sample positivity was made with primer set No. 1 and confirmed with either primer sets 2 or 3 which amplify nonoverlapping regions of the KSHV hypothetical major capsid gene. Sampling two portions of the KSHV genome decreased the likelihood of intraexperimental PCR contamination. These nested primer sets are 2-3 logs more sensitive for detecting KSHV sequences than the previously published KS330₂₃₃ primers [6] and are estimated to be able to detect <10 copies of KSHV genome under optimal conditions. Sample preparations were prealiquoted and amplified with alternating negative control samples without DNA to monitor and control possible contamination. All samples were tested in a blinded fashion and a determination of the positivity/negativity made before code breaking. Significance testing was performed with Mantel-Haenszel chi-squared estimates and exact confidence intervals using Epi-Info ver. 6 (USD Inc., Stone Mt. Ga.).

RESULTS

KSHV Positivity of Case and Control PBMC Samples:

Paired PBMC samples were available from each KS patient and homosexual/bisexual control patient; a single sample was available from each hemophilic control patient.

To determine the KSHV positivity rate for each group of AIDS patients, a single specimen from each participant taken closest to KS or other AIDS-defining illness ("second sample") was analyzed. Overall, 12 of 21 (57%) of PBMC specimens from KS patients taken from 6 months prior to KS diagnosis to 20 months after KS diagnosis were KSHV positive. There was no apparent difference in positivity rate between immediate pre-diagnosis and post-diagnosis visit specimens (4 of 7 (57%) vs. 8 of 14 (57%) respectively).

The number of KSHV positive control PBMC specimens from both homosexual/bisexual (second visit) and hemophilic patient controls was significantly lower. Only 2 of 19 (11%) hemophilic PBMC samples were positive (odds ratio 11.3, 95 % confidence interval 1.8 to 118) and only 2 of 23 (9%) PBMC samples from homosexual/bisexual men who did not develop KS were positive (odds ratio 14.0, 95% confidence interval 2.3 to 144). If all KS patient PBMC samples taken immediately prior to or after diagnosis were truly infected, the PCR assay was at least 57% sensitive in detecting KSHV infection among PBMC samples. No significant differences in CD4+ counts were found for KS patients and homosexual/bisexual patients without KS at the second sample evaluation (Kruskall-Wallis p=0.15) (FIG. 21). CD4+ counts from the single sample from hemophilic AIDS patients were higher than CD4+ counts from KS patients (Kruskall-Wallis p=0.004), although both groups showed evidence of HIV-related immunosuppression.

Longitudinal Studies:

Paired specimens were available from all 21 KS patients and 23 homosexual/bisexual male AIDS control patients who did not develop KS. For the KS group, initial PBMC samples were taken four to 87 months (median 13 months) prior to the onset of KS. Initial PBMC samples from the control group were drawn 13 to 106 months (median 55 months) prior to onset of first nonKS AIDS-defining illness (1987 CDC surveillance definition). 11 of 21 (52%) of KS patients had detectable KSHV DNA in PBMC samples taken prior to KS onset compared to 2 of 19 (11%, p=0.005) hemophilic control samples, and 1 (4%, p=0.0004) and 2 (9%, p=0.002) of 23 homosexual/bisexual control samples taken at the first and second visits respectively (FIGS. 20A-20B). The figure shows that 7 of the paired KS patient samples were positive at both visits, 5 KS patients and 2 control patients converted from negative to positive and two KS patients and one control patient reverted from positive to negative between visits. The remaining 7 KS patients and 20 control patients were negative at both visits.

For the 5 KS patients that converted from an initial negative PBMC result to a positive result at or near to KS diagnosis, the median length of time between the first sample and the KS diagnosis was 19 months. Three of the 6 KS patients that were negative at both visits had their last PBMC sample drawn 2-3 months prior to onset of illness. It is unknown whether these patients became infected between their last study visit and the KS diagnosis date.

DISCUSSION

Ambroziak and coworkers have found evidence that KSHV preferentially infects CD19+ B cells by PBMC subset examination of three patients [19]. Other gammaherpesviruses, such as Epstein-Barr virus (EBV) and herpesvirus saimiri are also lymphotrophic herpesviruses and can cause lymphoproliferative disorders in primates [11, 20].

It is possible that KSHV, like most human herpesviruses, is a ubiquitous infection of adults [21]. EBV, for example, is detectable by PCR in CD19+ B lymphocytes from virtually all seropositive persons [22] and approximately 98% MACS study participants had EBV VCA antibodies at entry into the cohort study [23]. The findings, however, are most consistent with control patients having lower KSHV infection rates than cases and that KSHV is specifically associated with the subsequent development of KS. While it is possible that control patients are infected but have an undetectably low KSHV viral PBMC load, the inability to find evidence of infection in control patients under a variety of PCR conditions suggests that the majority of control patients are not infected. Nonetheless, approximately 10% of these patients were KSHV infected and did not develop KS. It is unknown whether or not this is similar to the KSHV infection rate for the general human population.

This study demonstrates that KSHV infection is both strongly associated with KS and precedes onset of disease in the majority of patients. 57% of KS patients had detectable KSHV infection at their second follow-up visit (52% prior to the onset of KS] compared to only 9% of homosexual/bisexual (p=0.002) and 11% of hemophilic control patients (p=0.005). Despite similar CD4+ levels between homosexual/bisexual KS cases and controls, KSHV DNA positivity rates were significantly higher for cases at both the first (p=0.005) and second sample visits indicating that immunosuppression alone was not responsible for these elevated detection rates. It is also unlikely that KSHV simply colonizes existing KS lesions in AIDS patients since neither patient group had KS at the time the initial sample was obtained. Five KS patients and two homosexual/bisexual control patients converted from a negative to a positive, possibly due to new infection acquired during the study period.

The findings are in contrast to PCR detection of KSHV DNA in all 10 PBMC samples from KS patients by Ambroziak et al. [19]. It is possible that the assay was not sensitive enough to detect virus in all samples since it was required that each positive sample to bus repeatedly positive by two independent primers in blinded PCR assays. This appears unlikely, however, given the sensitivity of the PCR nested primer sets. The 7 KS patients who were persistently negative on both paired samples may represent an aviremic or low viral load subpopulation of KS patients. The PCR conditions test a DNA amount equivalent to approximately 2×10³ lymphocytes; an average viral load less than 1 copy per 2×10³ cells may be negative in the assay. Two KS patients and a homosexual/bisexual control patient initially positive for KSHV PCR amplification reverted to negative in samples drawn after diagnosis. These results probably reflect inability to detect KSHV DNA in peripheral blood rather than true loss of infection although more detailed studies of the natural history of infection are needed.

The study was designed to answer the fundamental question of whether or not infection with KSHV precedes development of the KS phenotype. The findings indicate that there is a strong antecedent association between KSHV infection and KS. This temporal relationship is an absolute requirement for establishing that KSHV is central to the causal pathway for developing KS. This study contributes additional evidence for a possible causal role for this virus in the development of KS.

REFERENCES

1. Katz MH, Hessol NA, Buchbinder SP, Hirozawa A, O'Malley P, Holmberg SD. Temporal trends of opportunistic infections and malignancies in homosexual men with AIDS. J Infect Dis. 1994;170:198-202.

2. Beral V, Peterman TA, Berkelman RL, Jaffe HW. Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet. 1990;335:123-128.

3. Archibald CP, Schechter MT, Le TN, Craib KJP, Montaner JSG, O'Shaughnessy MV. Evidence for a sexually transmitted cofactor for AIDS-related Kaposi's sarcoma in a cohort of homosexual men. Epidemiol. 1992;3:203-209.

4. Beral V, Bull D, Jaffe H, Evans B, Gill N, Tillett H et al. Is risk of Kaposi's sarcoma in AIDS patients in Britain increased if sexual partners came from United States or Africa? BMJ. 1991;302:624-5.

5. Beral V. Epidemiology of Kaposi's sarcoma. Cancer, HIV and AIDS. London: Imperial Cancer Research Fund; 1991:5-22.

6. Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 1994;265:1865-69.

7. Moore PS, Chang Y. Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. New England J Med. 1995;332:1181-1185.

8. Boshoff C, Whitby D, Hatziionnou T, Fisher C, van der Walt J, Hatzakis A et al. Kaposi's sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet. 1995;345:1043-44.

9. Su I-J, Hsu Y-S, Chang Y-C, Wang I-W. Herpesvirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet. 1995;345:722-23.

10. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin JT, Harvard S, et al. Herpesvirus-like DNA in patients with Mediterranean Kaposi's sarcoma. Lancet. 1995;345:761-2.

11. Miller G. Oncogenicity of Epstein-Barr virus. J Infect Dis. 1974;130:187-205.

12. Hill AB. Environment and disease: association or causation? Proc Roy Soc Med. 1965;58:295-300.

13. Susser M. Judgment and causal inference: criteria in epidemiologic studies. Am J Epid. 1977;105:1-15.

14. Fishbein DB, Kaplan JE, Spira TJ, Miller B, Schonberger LB, Pinsky PF, et al. Unexplained lymphadenopathy in homosexual men: a longitudinal study. JAMA. 1985;254:930-5.

15. Holmberg SD. Possible cofactors for the development of AIDS-related neoplasms. Cancer Detection and Prevention. 1990;14:331-336.

16. Kaslow RA, Ostrow DG, Detels R, Phair JP, Polk BF, Rinaldo CR. The Multicenter AIDS Cohort Study: rationale, organization and selected characteristics of the participants. Am J Epidemiol. 1987;126:310-318.

17. Wolinsky S, Rinaldo C, Kwok S, Sinsky J, Gupta P, Imagawa D, et al. Human immunodeficiency virus type 1 (HIV-1) infection a median of 18 months before a diagnostic Western blot. Ann Internal Med. 1989;111:961.

18. Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, et al. Genital papillomavirus infection in female university students as determined by a PCR-based method. JAMA. 1991;265:2809-10.

19. Ambroziak JA, Blackbourn DJ, Herndier BG, Glogau RG, Gullett JH, McDonald AR, et al. Herpes-like sequences in HIV-infected and uninfected Kaposi's sarcoma patients. Science. 1995;268:582-583.

20. Roizman B. The family Herpesviridae. In: Roizman B, Whitley RJ, Lopez C, eds. The Human Herpeviruses. New York: Raven Press, Ltd.; 1993:1-9.

21. Roizman B. New viral footprints in Kaposi's sarcoma. N Engl J Med. 1995;332:1227-1228.

22. Miyashita EM, Yang B, Lam KMC, Crawford DH, Thorley-Lawson DA. A novel form of Epstein-Barr virus latency in normal B cells in vivo. Cell. 1995;80:593-601.

23. Rinaldo CR, Kingsley LA, Lyter DW, Rabin BS, Atchison RW, Bodner AJ, et al. Association of HTLV-III with Epstein-Barr virus infection and abnormalities of T lymphocytes in homosexual men. J Infect Dis. 1986;154:556-61.

EXPERIMENTAL DETAILS SECTION IV

To determine if the KHV-KS virus is also present in both endemic and HIV-associated KS lesions from African patients, formalin-fixed, paraffin-embedded tissues from both HIV seropositive and HIV seropositive Ugandan KS patients were compared to cancer tissues from patients without KS in a blinded case-control study.

Patient Enrollment:

Archival KS biopsy specimens were selected from approximately equal numbers of HIV-associated and endemic HIV-negative KS patients enrolled in an ongoing case-control study of cancer and HIV infection at Makerere University, Kampala Uganda. Control tissues were consecutive archival biopsies from patients with various malignancies enrolled in the same study, chosen without prior knowledge of HIV serostatus. All patients were tested for HIV antibody (measured by Cambridge Bioscience Recombigen Elisa assay).

Tissue preparation:

Each sample examined was from an individual patient. Approximately ten tissue sections were cut (10 micron) from each paraffin block using a cleaned knife blade for each specimen. Tissue sections were deparaffinized by extracting the sections twice with 1 ml xylene for 15 min. followed by two extractions with 100% ethanol for 15 min. The remaining pellet was then resuspended and incubated overnight at 50° C. in 0.5 ml of lysis buffer (25 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.4 mM MgCl2, 0.01% gelatin, 1 mg/ml proteinase K). DNA was extracted with phenol/chloroform, ethanol precipitated and resuspended in 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.3.

PCR Amplification:

0.2-0.4 ug of DNA was used in PCR reactions with KS330₂₃₃ primers as previously described [7]. The samples which were negative were retested by nested PCR amplification, which is approximately 10² -10³ fold more sensitive in detecting KS330₂₃₃ sequence than the previously published KS330₂₃₃ primer set [7]. These samples were tested twice and samples showing discordant results were retested a third time. 51 of 74 samples initially examined were available for independent extraction and testing at Chester Beatty Laboratories, London using identical nested PCR primers and conditions to ensure fidelity of the PCR results. Results from eight samples were discordant between laboratories and were removed from the analysis as uninterpretable (four positive samples from each laboratory). Statistical comparisons were made using EPI-INFO ver. 5 (USD, Stone Mt. Ga., USA) with exact confidence intervals.

RESULTS

Of 66 tissues examined, 24 were from AIDS-KS cases, 20 were from endemic HIV seronegative KS cases, and 22 were from cancer control patients without KS. Seven of the cancer control patients were HIV seropositive and 15 were HIV seronegative (FIG. 22). Tumors examined in the control group included carcinomas of the breast, ovaries, rectum, stomach, and colon, fibrosarcoma, lymphocytic lymphomas, Hodgkin's lymphomas, choriocarcinoma and anaplastic carcinoma of unknown primary site. The median age of AIDS-KS patients was 29 years (range 3-50) compared to 36 years (range 3-79) for endemic KS patients and 38 years (range 21-73) for cancer controls.

Among KS lesions, 39 of 44 (89%) were positive for KS330₂₃₃ PCR product, including KS tissues from 22 of 24 (92%) HIV seropositive and 17 of 20 (85%) HIV seronegative patients. In comparison, 3 of 22 (14%) nonKS cancer control tissues were positive, including 1 of 7 (14%) HIV seropositive and 2 of 15 (13%) HIV seronegative control patients (FIG. 19). These control patients included a 73 year old HIV seronegative male and a 29 year old HIV seronegative female with breast carcinomas, and a 36 year old HIV seropositive female with ovarian carcinoma. The odds ratios for detecting the sequences in tissues from HIV seropositive and HIV seronegative cases and controls wag 66 (95% confidence interval (95% C.I.) 3.8-3161) and 36.8 (95% C.I. 4.3-428) respectively. The overall weighted Mantel-Haenzel odds ratio stratified by HIV serostatus was 49.2 (95% C.I. 9.1-335). KS tissues from four HIV seropositive children (ages 3, 5, 6, and 7 years) and four HIV seronegative children (ages 3, 4, 4, and 12 years) were all positive for KS330₂₃₃.

All discordant results (i.e. KSHV negative KS or KSHV positive nonKS cancers) were reviewed microscopically. All KS330₂₃₃ PCR negative KS samples were confirmed to be KS. Likewise, all KS330₂₃₃ PCR positive nonKS cancers were found not to have occult KS histopathologically.

DISCUSSION

These results indicate that KSHV DNA sequences are found not only in AIDS-KS [5], classical KS [6] and transplant KS [7] but also in African KS from both HIV seropositive and seronegative patients. Despite differences in clinical and epidemiological features, KSHV DNA sequences are present in all major clinical subtypes of KS from widely dispersed geographic settings.

This study was performed on banked, formalin-fixed tissues which prevented the use of specific detection assays such as Southern hybridization. DNA extracted after such treatment is often fragmented which reduces the detection sensitivity of PCR and may account for the 5 PCR negative KS samples found in the study. The results, however, are unlikely to be due to PCR contamination or nonspecific amplification. Specimens were tested blindly and a subset of samples were independently extracted and tested at a physically separate laboratory. Specimen blinding is essential to ensure the integrity of results based solely on PCR analyses. A subset of amplicons was sequenced and found to be more than 98% identical to the published KS330₂₃₃ sequence confirming their specific nature and, because of minor sequence variation, making the possibility of contamination unlikely.

In contrast to previous studies in North American and European populations, it was found 3 of 22 control tissues to have evidence of KSHV infection. Since these cancers represent a variety of tissue types, it is unlikely that KSHV has an etiologic role in these tumors. One possible explanation for the findings is that these results reflect the rate of KSHV infection in the nonKS population in Uganda. Four independent controlled studies from North America [5 and9 ] Europe [7] and Asia [8] have failed to detect evidence of KSHV infection in over 200 cancer control tissues, with the exception of an unusual AIDS-associated, body-cavity-based lymphoma [9]. Taken together, these studies indicate that DNA-based detection of KSHV infection is rare in most nonKS cancer tissues from developed countries. KSHV infection has been reported in post-transplant skin tumors, although well-controlled studies are needed to confirm that these findings are not due to PCR contamination [10]. Since the rate of HIV-negative KS is much more frequent in Uganda than the United States, detection of KSHV in control tissues from cancer patients in the study may reflect a relatively high prevalence infection in the general Ugandan population.

While KS is extremely rare among children in developed countries [2], the rate of KS in Ugandan children has risen dramatically over the past 3 decades: age-standardized rates (per 100,000) for boys age 0-14 years were 0.25 in 1964-68 and 10.1 in 1992-93. Detection of KSHV genome in KS lesions from prepubertal children suggests that the virus has a nonsexual mode of transmission among Ugandan children. That five of these children were 5 years old or less raises the possibility that the agent can be transmitted perinatally. Whether or not immune tolerance due to perinatal transmission accounts for the more fulminant form of KS occurring in African children remains to be investigated.

REFERENCES

1. Oettle A. G. Geographic and racial differences in the frequency of Kaposi's sarcoma as evidence of environmental or genetic causes. Acta Un Int Cancer 1962;18:330-363.

2. Beral V. Epidemiology of Kaposi's sarcoma. In: Cancer, HIV and AIDS. London: Imperial Cancer Research Fund, 1991: 5-22.

3. Wabinga H. R., Parkin D. M., Wabwire-Mangen F., Mugerwa J. Cancer in Kampala, Uganda, in 1989-91: changes in incidence in the era of AIDS. Int J Cancer 1993;54:26-36.

4. Kestens L. et al. Endemic Kaposi's sarcoma is not associated with immunodeficiency. Int. J. Cancer 1985;36:49-54.

5. Chang Y. et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 1994; 266:1865-9.

6. Moore P.S. and Chang Y. Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. New England J Med 1995; 332:1181-85.

7. Boshoff C. et al. Kaposi's sarcoma-associated herpesvirus in HIV negative Kaposi's sarcoma (letter). Lancet 1995; 345:1043-44.

8. Su, I.-J., Hsu, Y.-S., Chang, Y.-C., Wang, I.-W. Herpevirus-like DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 1995;345: 722-3.

9. Cesarman E., Chang Y., Moore P. S., Said J. W., Knowles D. M. Kaposi's sarcoma-associated herpesvirus-like DNA sequences are present in AIDS-related body cavity based lymphomas. New England J Med 1995; 332:1186-1191.

10. Rady P. L. , et al. Herpesvirus-like DNA sequences in nonKaposi's sarcoma skin lesions of transplant patients. Lancet 1995;345:1339-40.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - <160> NUMBER OF SEQ ID NOS: 58                                        - - <210> SEQ ID NO 1                                                         <211> LENGTH: 20710                                                            <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 1                                                          - - tcgagtcgga gagttggcac aggccttgag ctcgctgtga cgttctcacg gt -             #gttggttg     60                                                                  - - ggatcagctg gtgactcaga caagtcttga gctctacaac gtaacatacg gg -             #ctgatgcc    120                                                                  - - cacccgatac cagaattacg cagtcggcaa ttctgtgccc tagagtcacc tc -             #aaagaata    180                                                                  - - atctgtggtg tccaagggga gggttctggg gccggctact tagaaaccgc ca -             #tagatcgg    240                                                                  - - gcagggtgga gtacttgagg agccggcggt aggtggccag gtgggcccgg tt -             #acctgctc    300                                                                  - - ttttgcgtgc tgctggaagc ctgctcaggg atttcttaac ctcggcctcg gt -             #tggacgta    360                                                                  - - ccatggcaga aggcggtttt ggagcggact cggtggggcg cggcggagaa aa -             #ggcctctg    420                                                                  - - tgactagggg aggcaggtgg gacttgggga gctcggacga cgaatcaagc ac -             #ctccacaa    480                                                                  - - ccagcacgga tatggacgac ctccctgagg agaggaaacc actaacggga aa -             #gtctgtaa    540                                                                  - - aaacctcgta catatacgac gtgcccaccg tcccgaccag caagccgtgg ca -             #tttaatgc    600                                                                  - - acgacaactc cctctacgca acgcctaggt ttccgcccag acctctcata cg -             #gcaccctt    660                                                                  - - ccgaaaaagg cagcattttt gccagtcggt tgtcagcgac tgacgacgac tc -             #gggagact    720                                                                  - - acgcgccaat ggatcgcttc gccttccaga gccccagggt gtgtggtcgc cc -             #tccccttc    780                                                                  - - cgcctccaaa tcacccacct ccggcaacta ggccggcaga cgcgtcaatg gg -             #ggacgtgg    840                                                                  - - gctgggcgga tctgcaggga ctcaagagga ccccaaaggg atttttaaaa ac -             #atctacca    900                                                                  - - aggggggcag tctcaaagcc cgtggacgcg atgtaggtga ccgtctcagg ga -             #cggcggct    960                                                                  - - ttgcctttag tcctaggggc gtgaaatctg ccatagggca aaacattaaa tc -             #atggttgg   1020                                                                  - - ggatcggaga atcatcggcg actgctgtcc ccgtcaccac gcagcttatg gt -             #accggtgc   1080                                                                  - - acctcattag aacgcctgtg accgtggact acaggaatgt ttatttgctt ta -             #cttagagg   1140                                                                  - - gggtaatggg tgtgggcaaa tcaacgctgg tcaacgccgt gtgcgggatc tt -             #gccccagg   1200                                                                  - - agagagtgac aagttttccc gagcccatgg tgtactggac gagggcattt ac -             #agattgtt   1260                                                                  - - acaaggaaat ttcccacctg atgaagtctg gtaaggcggg agacccgctg ac -             #gtctgcca   1320                                                                  - - aaatatactc atgccaaaac aagttttcgc tccccttccg gacgaacgcc ac -             #cgctatcc   1380                                                                  - - tgcgaatgat gcagccctgg aacgttgggg gtgggtctgg gaggggcact ca -             #ctggtgcg   1440                                                                  - - tctttgatag gcatctcctc tccccagcag tggtgttccc tctcatgcac ct -             #gaagcacg   1500                                                                  - - gccgcctatc ttttgatcac ttctttcaat tactttccat ctttagagcc ac -             #agaaggcg   1560                                                                  - - acgtggtcgc cattctcacc ctctccagcg ccgagtcgtt gcggcgggtc ag -             #ggcgaggg   1620                                                                  - - gaagaaagaa cgacgggacg gtggagcaaa actacatcag agaattggcg tg -             #ggcttatc   1680                                                                  - - acgccgtgta ctgttcatgg atcatgttgc agtacatcac tgtggagcag at -             #ggtacaac   1740                                                                  - - tatgcgtaca aaccacaaat attccggaaa tctgcttccg cagcgtgcgc ct -             #ggcacaca   1800                                                                  - - aggaggaaac tttgaaaaac cttcacgagc agagcatgct acctatgatc ac -             #cggtgtac   1860                                                                  - - tggatcccgt gagacatcat cccgtcgtga tcgagctttg cttttgtttc tt -             #cacagagc   1920                                                                  - - tgagaaaatt acaatttatc gtagccgacg cggataagtt ccacgacgac gt -             #atgcggcc   1980                                                                  - - tgtggaccga aatctacagg cagatcctgt ccaatccggc tattaaaccc ag -             #ggccatca   2040                                                                  - - actggccagc attagagagc cagtctaaag cagttaatca cctagaggag ac -             #atgcaggg   2100                                                                  - - tctagccttc ttggcggccc ttgcatgctg gcgatgcata tcgttgacat gt -             #ggagccac   2160                                                                  - - tggcgcgttg ccgacaacgg cgacgacaat aacccgctcc gccacgcagc tc -             #atcaatgg   2220                                                                  - - gagaaccaac ctctccatag aactggaatt caacggcact agtttttttc ta -             #aattggca   2280                                                                  - - aaatctgttg aatgtgatca cggagccggc cctgacagag ttgtggacct cc -             #gccgaagt   2340                                                                  - - cgccgaggac ctcagggtaa ctctgaaaaa gaggcaaagt ctttttttcc cc -             #aacaagac   2400                                                                  - - agttgtgatc tctggagacg gccatcgcta tacgtgcgag gtgccgacgt cg -             #tcgcaaac   2460                                                                  - - ttataacatc accaagggct ttaactatag cgctctgccc gggcaccttg gc -             #ggatttgg   2520                                                                  - - gatcaacgcg cgtctggtac tgggtgatat cttcgcatca aaatggtcgc ta -             #ttcgcgag   2580                                                                  - - ggacacccca gagtatcggg tgttttaccc aatgaatgtc atggccgtca ag -             #ttttccat   2640                                                                  - - atccattggc aacaacgagt ccggcgtagc gctctatgga gtggtgtcgg aa -             #gatttcgt   2700                                                                  - - ggtcgtcacg ctccacaaca ggtccaaaga ggctaacgag acggcgtccc at -             #cttctgtt   2760                                                                  - - cggtctcccg gattcactgc catctctgaa gggccatgcc acctatgatg aa -             #ctcacgtt   2820                                                                  - - cgcccgaaac gcaaaatatg cgctagtggc gatcctgcct aaagattctt ac -             #cagacact   2880                                                                  - - ccttacagag aattacactc gcatatttct gaacatgacg gagtcgacgc cc -             #ctcgagtt   2940                                                                  - - cacgcggacg atccagacca ggatcgtatc aatcgaggcc aggcgcgcct gc -             #gcagctca   3000                                                                  - - agaggcggcg ccggacatat tcttggtgtt gtttcagatg ttggtggcac ac -             #tttcttgt   3060                                                                  - - tgcgcggggc attgccgagc accgatttgt ggaggtggac tgcgtgtgtc gg -             #cagtatgc   3120                                                                  - - ggaactgtat tttctccgcc gcatctcgcg tctgtgcatg cccacgttca cc -             #actgtcgg   3180                                                                  - - gtataaccac accacccttg gcgctgtggc cgccacacaa atagctcgcg tg -             #tccgccac   3240                                                                  - - gaagttggcc agtttgcccc gctcttccca ggaaacagtg ctggccatgg tc -             #cagcttgg   3300                                                                  - - cgcccgtgat ggcgccgtcc cttcctccat tctggagggc attgctatgg tc -             #gtcgaaca   3360                                                                  - - tatgtatacc gcctacactt atgtgtacac actcggcgat actgaaagaa aa -             #ttaatgtt   3420                                                                  - - ggacatacac acggtcctca ccgacagctg cccgcccaaa gactccggag ta -             #tcagaaaa   3480                                                                  - - gctactgaga acatatttga tgttcacatc aatgtgtacc aacatagagc tg -             #ggcgaaat   3540                                                                  - - gatcgcccgc ttttccaaac cggacagcct taacatctat agggcattct cc -             #ccctgctt   3600                                                                  - - tctaggacta aggtacgatt tgcatccagc caagttgcgc gccgaggcgc cg -             #cagtcgtc   3660                                                                  - - cgctctgacg cggactgccg ttgccagagg aacatcggga ttcgcagaat tg -             #ctccacgc   3720                                                                  - - gctgcacctc gatagcttaa atttaattcc ggcgattaac tgttcaaaga tt -             #acagccga   3780                                                                  - - caagataata gctacggtac ccttgcctca cgtcacgtat atcatcagtt cc -             #gaagcact   3840                                                                  - - ctcgaacgct gttgtctacg aggtgtcgga gatcttcctc aagagtgcca tg -             #tttatatc   3900                                                                  - - tgctatcaaa cccgattgct ccggctttaa cttttctcag attgataggc ac -             #attcccat   3960                                                                  - - agtctacaac atcagcacac caagaagagg ttgccccctt tgtgactctg ta -             #atcatgag   4020                                                                  - - ctacgatgag agcgatggcc tgcagtctct catgtatgtc actaatgaaa gg -             #gtgcagac   4080                                                                  - - caacctcttt ttagataagt cacctttctt tgataataac aacctacaca tt -             #cattattt   4140                                                                  - - gtggctgagg gacaacggga ccgtagtgga gataaggggc atgtatagaa ga -             #cgcgcagc   4200                                                                  - - cagtgctttg tttctaattc tctcttttat tgggttctcg ggggttatct ac -             #tttcttta   4260                                                                  - - cagactgttt tccatccttt attagacggt caataaagcg tagattttta aa -             #aggtttcc   4320                                                                  - - tgtgcattct ttttgtatgg gcatatactt ggcaagaaat ccgagcacct ca -             #gaaagtgg   4380                                                                  - - attgccgtca catatcagtt cgaccacccc tgcacctagc catgcggcgc tt -             #tgacggtc   4440                                                                  - - tttggggcta cacatcataa agtacttttc catggcttct ataagcacct tg -             #gaacaatc   4500                                                                  - - tgggggttgg cgaatgggtt ccctaaacgg gaaatcctct atggtattca gg -             #cagaagac   4560                                                                  - - cgcgtcctcc acccgacgtt tgagtctttc tagcagagcg ccgaagaact cc -             #cgctcgtg   4620                                                                  - - tgttttcgca ggggcaagtt ctgcgccgta cagcgatgag aaacacgaca cg -             #atgttttc   4680                                                                  - - cagccccatg ctgcgcagca acacgtgctt caggaacagg tgttgtagcc gg -             #ttcagttt   4740                                                                  - - tagcttgggt agaaaagtta tcgagttgtt agcacgctcc atgatggtaa cg -             #gtgttgaa   4800                                                                  - - gtcacagacc gggctttctc cgagtctcgg ccgcctgagt ccaatcatgt ag -             #aacataga   4860                                                                  - - cgcggcctcg ttgtctgtgt taagtgacac gatatcccgt tcgcaaacct gt -             #gcgatgtt   4920                                                                  - - gtgtttcagt atagatctgg tctgaccggc acggggtgtt atggggtgac gc -             #ggtaaagg   4980                                                                  - - cgactctggg tcaaacacct ttatgcggtt ggcggcctcg tcgatgacga ca -             #cgcttgtt   5040                                                                  - - cgcggcgtgt atggggacgc gacggcatcc cgctggcaga tctataatct ta -             #aagttggt   5100                                                                  - - ataagactgg tcgctcgtta tggccagccg gcactccggt agtatctgcg tg -             #tcctcgaa   5160                                                                  - - ttcgtggccg cgtacgactg gcttggagtg caggtaaacg ccaagagatg cg -             #gtctcttc   5220                                                                  - - gcctacgcac aagtggcttc ttaacgcgta ggggtgcggt gagagcatga tc -             #cgtagcaa   5280                                                                  - - cgatagttcc gggtgcctag ccgcgtagag tggcagggta gacgagtccg ga -             #gtcccaaa   5340                                                                  - - cttttcgaac aacagtggca tcgggacttc aggattagag actcccacca tg -             #gccgccac   5400                                                                  - - cgccggagag gtcaagacgt gaaacacgcg ctcgcctgtc gacaggcgcg cc -             #gcgccctc   5460                                                                  - - tactagacta gccttcacgt ccggaactcg taacatagct tagaccagcg ga -             #cggacgca   5520                                                                  - - acgtacgcgg ggatcggctg gcggtgtctg ctcgttggac gcggccgttc gg -             #tggcgcca   5580                                                                  - - gtgcaggcct agtttgcgaa tggcgtgacg gacaatttgt ggctttagag cg -             #gcgaaccg   5640                                                                  - - atgacccgtg gtggcgacga acgaaatgaa gtttgcattg cggcccaact cg -             #tctagcct   5700                                                                  - - ggtcttcttg tttcgggcat agattttcgg gattaggtta cactttttat at -             #cccagtac   5760                                                                  - - tgcgcactcg tgtttgcttt tagtgtgact gattatcttc tttgagaagt ca -             #aacaggcc   5820                                                                  - - ccgggcggcg gctcgcctaa tgcaagccac gtcaagcctg agaaacgaac ag -             #cattccac   5880                                                                  - - cagacactcc aggaaccttt tgtgtagcgt ctgtatttgg gaacggtttc tg -             #tgctcaag   5940                                                                  - - tagggagaat attctatttt tgtttccgtc gatgcgcgcg tgctggtccg tg -             #agaatggg   6000                                                                  - - cgccagctcg tggcgaatct gttccacaag aggctgcccg tacactttag aa -             #atcgtggc   6060                                                                  - - tgtcgcggcc ttaaaccagg acacgtttag cccatccttg ctggagacca ca -             #gatggaaa   6120                                                                  - - gtttgtggtc caaaatacgt tttttcgccc cattctcacc atgtactggt tt -             #tccagtcc   6180                                                                  - - gtgcaggtcc aacgtggagt tccaatttgc tatcgataca ggaaatatgt gc -             #ctgattgg   6240                                                                  - - cagaaagcat ttcagcgtac ccattgcgaa gagaaagtgc agcatgtccc ca -             #ctgatgtt   6300                                                                  - - gatgtttatt gcggtgcctt gacacatgtt gtcggaaaaa aacacgctta tg -             #gtaaaaga   6360                                                                  - - aggttccttt acggagtact ttcgtataac aaaattgttg gtcaatctgg gg -             #atgtttaa   6420                                                                  - - aatagtcttt tgcagggtgt taggaacgtg gcagcttatc ttagtgttaa tc -             #accatgtt   6480                                                                  - - ggtgttgaat atggtgatct tgaagttttc caaactgacg tgttttgtgg gt -             #tccagcat   6540                                                                  - - gtctgacact gtagagctgc ccagagtccg cgcgtccgtg gccgcgtatc gt -             #tggaagca   6600                                                                  - - cgcctgcaaa tttcctttca tggctgctcg ccggtctttc ggcgcgtacc gg -             #attcttga   6660                                                                  - - aagcgtcgcc gccaggagac gcggtgtctc gtgggtgcct aaaaagtttg cg -             #caggggtg   6720                                                                  - - cagtccgctg cacgagtggc cgatgcagtc tgccactgcc atacacatga cg -             #agtctgta   6780                                                                  - - gatggccggt gtgcccggat acactagata gtaggtacaa tctggggtac tg -             #acgaccac   6840                                                                  - - cctgtatggc tttggtccgg ggtccttgcg ttggattttt acgtgcagac gg -             #gacacgag   6900                                                                  - - ctggtttaga gccagctgaa agcccaccag atcccgtccg ttaaccttga cg -             #tcctggtg   6960                                                                  - - cttactctgt ttcgacaggt tcttcagcac ggtgggcagt cgctctacgt tg -             #tgagcgat   7020                                                                  - - ggcacggcgc agcgagacca gctctccgtg ccacccccac gtggccatga ag -             #ctgctgat   7080                                                                  - - gttaaacttt aaaaaatgta gctgtgcgtc tggggatgcg ggtggcatta tt -             #gaaaacga   7140                                                                  - - gagatgcttc aggctctcca ggagtgcaaa ataattttga tagattgtgg gt -             #tgtagact   7200                                                                  - - atggggcaac accgccagaa acgcatgaaa acactgttcg aactcccaga ac -             #tccaggta   7260                                                                  - - cctgcacact atcctgaaca tggctttgta acatatggtg cacgttagta gc -             #gcgggaag   7320                                                                  - - atacagcgag cgtagctccc tgaattcgca gggtttatca caatcatcgg ta -             #agttccca   7380                                                                  - - tgatcccacc gcaggtaggt agttgtcggt gtctatctgt ccgcgcgtaa ac -             #actccacc   7440                                                                  - - accgtcaatt attaaacctt cgccgctgta ccgtcgaccc acttttccca aa -             #agagtccc   7500                                                                  - - ttcttgatgt ataaaagggt ggaggcgttc ccccaggagt agtctgcgta tc -             #gctctgca   7560                                                                  - - ggcgaaaaag gtgggctcgg gctgcatcat cttatcaaga ccttctaagg tc -             #agctctgc   7620                                                                  - - ctgcaggtgc gagttggtgg ccagacagca gaatatttcc agctgtgatt cc -             #caagtcgc   7680                                                                  - - ttgataacac gtggtctgcg gactcgtcgt cagggaggcg ctcggtggca gt -             #agtagggg   7740                                                                  - - gccctcgagc gctgccatgg aggcgacctt ggagcaacga cctttcccgt ac -             #ctcgccac   7800                                                                  - - ggaggccaac ctcctaacgc agattaagga gtcggctgcc gacggactct tc -             #aagagctt   7860                                                                  - - tcagctattg ctcggcaagg acgccagaga aggcagtgtc cgtttcgaag cg -             #ctactggg   7920                                                                  - - cgtatatacc aatgtggtgg agtttgttaa gtttctggag accgccctcg cc -             #gccgcttg   7980                                                                  - - cgtcaatacc gagttcaagg acctgcggag aatgatagat ggaaaaatac ag -             #tttaaaat   8040                                                                  - - ttcaatgccc actattgccc acggagacgg gaggaggccc aacaagcaga ga -             #cagtatat   8100                                                                  - - cgtcatgaag gcttgcaata agcaccacat cggtgcggag attgagcttg cg -             #gccgcaga   8160                                                                  - - catcgagctt ctcttcgccg agaaagagac gcccttggac ttcacagagt ac -             #gcgggtgc   8220                                                                  - - catcaagacg attacgtcgg ctttgcagtt tggtatggac gccctagaac gg -             #gggctagt   8280                                                                  - - ggacacggtt ctcgcagtta aacttcggca cgctccaccc gtctttattt ta -             #aagacgct   8340                                                                  - - gggcgatccc gtctactctg agaggggcct caaaaaggcc gtcaagtctg ac -             #atggtatc   8400                                                                  - - catgttcaag gcacacctca tagaacattc attttttcta gataaggccg ag -             #ctcatgac   8460                                                                  - - aagggggaag cagtatgtcc taaccatgct ctccgacatg ctggccgcgg tg -             #tgcgagga   8520                                                                  - - taccgtcttt aagggtgtca gcacgtacac cacggcctct gggcagcagg tg -             #gccggcgt   8580                                                                  - - cctggagacg acggacagcg tcatgagacg gctgatgaac ctgctggggc aa -             #gtggaaag   8640                                                                  - - tgccatgtcc gggcccgcgg cctacgccag ctacgttgtc aggggtgcca ac -             #ctcgtcac   8700                                                                  - - cgccgttagc tacggaaggg cgatgagaaa ctttgaacag tttatggcac gc -             #atagtgga   8760                                                                  - - ccatcccaac gctctgccgt ctgtggaagg tgacaaggcc gctctggcgg ac -             #ggacacga   8820                                                                  - - cgagattcag agaacccgca tcgccgcctc tctcgtcaag ataggggata ag -             #tttgtggc   8880                                                                  - - cattgaaagt ttgcagcgca tgtacaacga gactcagttt ccctgcccac tg -             #aaccggcg   8940                                                                  - - catccagtac acctatttct tccctgttgg ccttcacctt cccgtgcccc gc -             #tactcgac   9000                                                                  - - atccgtctca gtcaggggcg tagaatcccc ggccatccag tcgaccgaga cg -             #tgggtggt   9060                                                                  - - taataaaaac aacgtgcctc tttgcttcgg ttaccaaaac gccctcaaaa gc -             #atatgcca   9120                                                                  - - ccctcgaatg cacaacccca cccagtcagc ccaggcacta aaccaagctt tt -             #cccgatcc   9180                                                                  - - cgacggggga catgggtacg gtctcaggta tgagcagacg ccaaacatga ac -             #ctattcag   9240                                                                  - - aacgttccac cagtattaca tggggaaaaa cgtggcattt gttcccgatg tg -             #gcccaaaa   9300                                                                  - - agcgctcgta accacggagg atctactgca cccaacctct caccgtctcc tc -             #agattgga   9360                                                                  - - ggtccacccc ttctttgatt tttttgtgca cccctgtcct ggagcgagag ga -             #tcgtaccg   9420                                                                  - - cgccacccac agaacaatgg ttggaaatat accacaaccg ctcgctccaa gg -             #gagtttca   9480                                                                  - - ggaaagtaga ggggcgcagt tcgacgctgt gacgaatatg acacacgtca ta -             #gaccagct   9540                                                                  - - aactattgac gtcatacagg agacggcatt tgaccccgcg tatcccctgt tc -             #tgctatgt   9600                                                                  - - aatcgaagca atgattcacg gacaggaaga aaaattcgtg atgaacatgc cc -             #ctcattgc   9660                                                                  - - cctggtcatt caaacctact gggtcaactc gggaaaactg gcgtttgtga ac -             #agttatca   9720                                                                  - - catggttaga ttcatctgta cgcatattgg gaatggaagc atccctaagg ag -             #gcgcacgg   9780                                                                  - - ccactaccgg aaaatcttag gcgagctcat cgcccttgag caggcgcttc tc -             #aagctcgc   9840                                                                  - - gggacacgag acggtgggtc ggacgccgat cacacatctg gtttcggctc tc -             #ctcgaccc   9900                                                                  - - gcatctgctg cctccctttg cctaccacga tgtctttacg gatcttatgc ag -             #aagtcatc   9960                                                                  - - cagacaaccc ataatcaaga tcggggatca aaactacgac aaccctcaaa at -             #agggcgac  10020                                                                  - - attcatcaac ctcaggggtc gcatggagga cctagtcaat aaccttgtta ac -             #atttacca  10080                                                                  - - gacaagggtc aatgaggacc atgacgagag acacgtcctg gacgtggcgc cc -             #ctggacga  10140                                                                  - - gaatgactac aacccggtcc tcgagaagct attctactat gttttaatgc cg -             #gtgtgcag  10200                                                                  - - taacggccac atgtgcggta tgggggtcga ctatcaaaac gtggccctga cg -             #ctgactta  10260                                                                  - - caacggcccc gtctttgcgg acgtcgtgaa cgcacaggat gatattctac tg -             #cacctgga  10320                                                                  - - gaacggaacc ttgaaggaca ttctgcaggc aggcgacata cgcccgacgg tg -             #gacatgat  10380                                                                  - - cagggtgctg tgcacctcgt ttctgacgtg ccctttcgtc acccaggccg ct -             #cgcgtgat  10440                                                                  - - cacaaagcgg gacccggccc agagttttgc cacgcacgaa tacgggaagg at -             #gtggcgca  10500                                                                  - - gaccgtgctt gttaatggct ttggtgcgtt cgcggtggcg gaccgctctc gc -             #gaggcggc  10560                                                                  - - ggagactatg ttttatccgg taccctttaa caagctctac gctgacccgt tg -             #gtggctgc  10620                                                                  - - cacactgcat ccgctcctgc caaactatgt caccaggctc cccaaccaga ga -             #aacgcggt  10680                                                                  - - ggtctttaac gtgccatcca atctcatggc agaatatgag gaatggcaca ag -             #tcgcccgt  10740                                                                  - - cgcggcgtat gccgcgtctt gtcaggccac cccgggcgcc attagcgcca tg -             #gtgagcat  10800                                                                  - - gcaccaaaaa ctatctgccc ccagtttcat ttgccaggca aaacaccgca tg -             #caccctgg  10860                                                                  - - ttttgccatg acagtcgtca ggacggacga ggttctagca gagcacatcc ta -             #tactgctc  10920                                                                  - - cagggcgtcg acatccatgt ttgtgggctt gccttcggtg gtacggcgcg ag -             #gtacgttc  10980                                                                  - - ggacgcggtg acttttgaaa ttacccacga gatcgcttcc ctgcacaccg ca -             #cttggcta  11040                                                                  - - ctcatcagtc atcgccccgg cccacgtggc cgccataact acagacatgg ga -             #gtacattg  11100                                                                  - - tcaggacctc tttatgattt tcccagggga cgcgtatcag gaccgccagc tg -             #catgacta  11160                                                                  - - tatcaaaatg aaagcgggcg tgcaaaccgg ctcaccggga aacagaatgg at -             #cacgtggg  11220                                                                  - - atacactgct ggggttcctc gctgcgagaa cctgcccggt ttgagtcatg gt -             #cagctggc  11280                                                                  - - aacctgcgag ataattccca cgccggtcac atctgacgtt gcctatttcc ag -             #acccccag  11340                                                                  - - caacccccgg gggcgtgcgg cgtcggtcgt gtcgtgtgat gcttacagta ac -             #gaaagcgc  11400                                                                  - - agagcgtttg ttctacgacc attcaatacc agaccccgcg tacgaatgcc gg -             #tccaccaa  11460                                                                  - - caacccgtgg gcttcgcagc gtggctccct cggcgacgtg ctatacaata tc -             #acctttcg  11520                                                                  - - ccagactgcg ctgccgggca tgtacagtcc ttgtcggcag ttcttccaca ag -             #gaagacat  11580                                                                  - - tatgcggtac aatagggggt tgtacacttt ggttaatgag tattctgcca gg -             #cttgctgg  11640                                                                  - - ggcccccgcc accagcacta cagacctcca gtacgtcgtg gtcaacggta ca -             #gacgtgtt  11700                                                                  - - tttggaccag ccttgccata tgctgcagga ggcctatccc acgctcgccg cc -             #agccacag  11760                                                                  - - agttatgctt gccgagtaca tgtcaaacaa gcagacacac gccccagtac ac -             #atgggcca  11820                                                                  - - gtatctcatt gaagaggtgg cgccgatgaa gagactatta aagctcggaa ac -             #aaggtggt  11880                                                                  - - gtattagcta acccttctag cgttggctag tcatggcact cgacaagagt at -             #agtggtta  11940                                                                  - - acttcacctc cagactcttc gctgatgaac tggccgccct tcagtcaaaa at -             #agggagcg  12000                                                                  - - tactgccgct cggagattgc caccgtttac aaaatataca ggcattgggc ct -             #ggggtgcg  12060                                                                  - - tatgctcacg tgagacatct ccggactaca tccaaattat gcagtatcta tc -             #caagtgca  12120                                                                  - - cactcgctgt cctggaggag gttcgcccgg acagcctgcg cctaacgcgg at -             #ggatccct  12180                                                                  - - ctgacaacct tcagataaaa aacgtatatg cccccttttt tcagtgggac ag -             #caacaccc  12240                                                                  - - agctagcagt gctaccccca ttttttagcc gaaaggattc caccattgtg ct -             #cgaatcca  12300                                                                  - - acggatttga ccccgtgttc cccatggtcg tgccgcagca actggggcac gc -             #tattctgc  12360                                                                  - - agcagctgtt ggtgtaccac atctactcca aaatatcggc cggggccccg ga -             #tgatgtaa  12420                                                                  - - atatggcgga acttgatcta tataccacca atgtgtcatt tatggggcgc ac -             #atatcgtc  12480                                                                  - - tggacgtaga caacacggat ccacgtactg ccctgcgagt gcttgacgat ct -             #gtccatgt  12540                                                                  - - acctttgtat cctatcagcc ttggttccca gggggtgtct ccgtctgctc ac -             #ggcgctcg  12600                                                                  - - tgcggcacga caggcatcct ctgacagagg tgtttgaggg ggtggtgcca ga -             #tgaggtga  12660                                                                  - - ccaggataga tctcgaccag ttgagcgtcc cagatgacat caccaggatg cg -             #cgtcatgt  12720                                                                  - - tctcctatct tcagagtctc agttctatat ttaatcttgg ccccagactg ca -             #cgtgtatg  12780                                                                  - - cctactcggc agagactttg gcggcctcct gttggtattc cccacgctaa cg -             #atttgaag  12840                                                                  - - cggggggggt atggcgtcat ctgatattct gtcggttgca aggacggatg ac -             #ggctccgt  12900                                                                  - - ctgtgaagtc tccctgcgtg gaggtaggaa aaaaactacc gtctacctgc cg -             #gacactga  12960                                                                  - - accctgggtg gtagagaccg acgccatcaa agacgccttc ctcagcgacg gg -             #atcgtgga  13020                                                                  - - tatggctcga aagcttcatc gtggtgccct gccctcaaat tctcacaacg gc -             #ttgaggat  13080                                                                  - - ggtgcttttt tgttattgtt acttgcaaaa ttgtgtgtac ctagccctgt tt -             #ctgtgccc  13140                                                                  - - ccttaatcct tacttggtaa ctccctcaag cattgagttt gccgagcccg tt -             #gtggcacc  13200                                                                  - - tgaggtgctc ttcccacacc cggctgagat gtctcgcggt tgcgatgacg cg -             #attttctg  13260                                                                  - - taaactgccc tataccgtgc ctataatcaa caccacgttt ggacgcattt ac -             #ccgaactc  13320                                                                  - - tacacgcgag ccggacggca ggcctacgga ttactccatg gcccttagaa gg -             #gcttttgc  13380                                                                  - - agttatggtt aacacgtcat gtgcaggagt gacattgtgc cgcggagaaa ct -             #cagaccgc  13440                                                                  - - atcccgtaac cacactgagt gggaaaatct gctggctatg ttttctgtga tt -             #atctatgc  13500                                                                  - - cttagatcac aactgtcacc cggaagcact gtctatcgcg agcggcatct tt -             #gacgagcg  13560                                                                  - - tgactatgga ttattcatct ctcagccccg gagcgtgccc tcgcctaccc ct -             #tgcgacgt  13620                                                                  - - gtcgtgggaa gatatctaca acgggactta cctagctcgg cctggaaact gt -             #gacccctg  13680                                                                  - - gcccaatcta tccacccctc ccttgattct aaattttaaa taaaggtgtg tc -             #actggtta  13740                                                                  - - caccacgatt aaaaaccact cactgagatg tctttttaac cgctaaggga tt -             #ataccggg  13800                                                                  - - atttaaaacc gcccactgat ttttttacgc taagagttgg gtgcttgggg gg -             #ttttgcat  13860                                                                  - - tgctctgttg taaactatat ataagttaaa ccaaaattcg cagggagaca ag -             #gtgacggt  13920                                                                  - - ggtgagaact cagttgagag tcagagaata cagtgctaat cagggtagat ga -             #gcatgact  13980                                                                  - - ttccccgtct ccagtcaccg gaggaatggt ggacggctcc gtcctggtgc ga -             #atggccac  14040                                                                  - - caagcctccc gtgattggtc ttataacagt gctcttcctc ctagtcatag gc -             #gcctgcgt  14100                                                                  - - ctactgctgc attcgcgtgt tcctggcggc tcgactgtgg cgcgccaccc ca -             #ctaggcag  14160                                                                  - - ggccaccgtg gcgtatcagg tccttcgcac cctgggaccg caggccgggt ca -             #catgcacc  14220                                                                  - - gccgacggtg ggcatagcta cccaggagcc ctaccgtaca atatacatgc ca -             #gattagaa  14280                                                                  - - cggggtgtgt gctataatgg atggctatgg gggggggctg tagataattg ag -             #cgctgtgc  14340                                                                  - - ttttattgtg gggatatggg cttgtacatg tgtctatcat cggtagccat aa -             #aatgggcc  14400                                                                  - - atgacaactg ccacaagtaa gtcgtccgac atgtgctttt gcttggcgct gt -             #atgactgc  14460                                                                  - - cctccatccc taagcgggac gcacttgatc gcgcggacct gttctaccag gt -             #aggtcacc  14520                                                                  - - gggtcaaatg atattttgat ggtgttggac accaccgtct ggctggcgct ca -             #gggtgccg  14580                                                                  - - gagttcagag cgtagatgaa tgtctcaaac gcggaggatt tctcgcctcc ca -             #acatgtaa  14640                                                                  - - attggccact gcagggcgct gctcttgtca gtatagtgta gaaaatgtat gg -             #ggagcggg  14700                                                                  - - catatttcgt taaggacggt tgcaatggcc accccagaat cttggctgct gt -             #tgccttcg  14760                                                                  - - accgccgcgt tcacgcgctc aattgtggtg tggagcacag cgatcgcctt aa -             #tcatcgtg  14820                                                                  - - catgcgcagg acgctatctc gtaagcagct gcgccagtga ggtcgcgcag ga -             #agaaatgc  14880                                                                  - - tccatgccca atatgaggct tctggtggga gtctgagtac tcgtgacaac gg -             #cgcccacg  14940                                                                  - - ccagtaccgg acgcctccgt gttgttcgta tacgcggggt cgatgtaaac aa -             #acagctgt  15000                                                                  - - tttccaaggc acttctgaac ctcctgggcg gtggtgtcta cccgacacat gt -             #caaactgt  15060                                                                  - - gtcagcgctg cgtcacccac cacgcggtaa agcgtagcat ttgacgacgc tg -             #ctccctcg  15120                                                                  - - cccattagtt cggtgtcgaa tgccccctcc ataaagaggt tggtggtggt tt -             #tgatggat  15180                                                                  - - tcgtcgatgg tgatgtacgt cggaatgtgc agtctgtaac aaggacagga ca -             #ctagtgcg  15240                                                                  - - tcttgcaggt ggaaatcttc tcggtggtcc gcacacacgt aactgaccac at -             #tcagcatc  15300                                                                  - - ttttcctggg cgttcctgag gttaagcagg aaactcgtgg agcggtctga cg -             #agttcacg  15360                                                                  - - gatgatataa atataagctt ggcgtctttc tgaagcatga aacccagaat ag -             #ccggcagt  15420                                                                  - - gcatcctttt taataaaatt cgcctcgtct acgtagagca ggttaaaggt ct -             #gtccccga  15480                                                                  - - atgctctgca gacacggaaa gacacaaaag aggggctcat aagcggctaa ca -             #gtaaagga  15540                                                                  - - gaggaggcga acagtgcgtg gctcttggtt cttgggaata aaagggggcg tg -             #tgtgccga  15600                                                                  - - tcgatcgtat gggtgagcca gtggatcctg gacatgtggt gaatgagaaa ga -             #ttttgagg  15660                                                                  - - agtgtgaaca atttttcagt caacccctta gggagcaagt ggtcgcgggg gt -             #cagggcac  15720                                                                  - - tcgacggcct cggtctcgct gactctctat gtcacaaaac agaaagactc tg -             #cctgctga  15780                                                                  - - tggacctggt gggcacggag tgctttgcga gggtgtgccg cctagacacc gg -             #tgcgaaat  15840                                                                  - - gaagagtgtg gcgagtccct tatgtcagtt ccacggcgtg ttttgcctgt ac -             #cagtgtcg  15900                                                                  - - ccagtgcctg gcataccacg tgtgtgatgg gggcgccgaa tgcgttctcc tg -             #catacgcc  15960                                                                  - - ggagagcgtc atctgcgaac taacgggtaa ctgcatgctc ggcaacattc aa -             #gagggcca  16020                                                                  - - gtttttaggg ccggtaccgt atcggacttt ggataaccag gttgacaggg ac -             #gcatatca  16080                                                                  - - cgggatgcta gcgtgtctga aacgggacat tgtgcggtat ttgcagacat gg -             #ccggacac  16140                                                                  - - caccgtaatc gtgcaggaaa tagccctggg ggacggcgtc accgacacca tc -             #tcggccat  16200                                                                  - - tatagatgaa acattcggtg agtgtcttcc cgtactgggg gaggcccaag gc -             #gggtacgc  16260                                                                  - - cctggtctgt agcatgtatc tgcacgttat cgtctccatc tattcgacaa aa -             #acggtgta  16320                                                                  - - caacagtatg ctatttaaat gcacaaagaa taaaaagtac gactgcattg cc -             #aagcgggt  16380                                                                  - - gcggacaaaa tggatgcgca tgctatcaac gaaagatacg taggtcctcg ct -             #gccaccgt  16440                                                                  - - ttggcccacg tggtgctgcc taggaccttt ctgctgcatc acgccatacc cc -             #tggagccc  16500                                                                  - - gagatcatct tttccaccta cacccggttc agccggtcgc cagggtcatc cc -             #gccggttg  16560                                                                  - - gtggtgtgtg ggaaacgtgt cctgccaggg gaggaaaacc aacttgcgtc tt -             #caccttct  16620                                                                  - - ggtttggcgc ttagcctgcc tctgttttcc cacgatggga actttcatcc at -             #ttgacatc  16680                                                                  - - tcggtactgc gcatttcctg ccctggttct aatcttagtc ttactgtcag at -             #ttctctat  16740                                                                  - - ctatctctgg tggtggctat gggggcggga cggaataatg cgcggagtcc ga -             #ccgttgac  16800                                                                  - - ggggtatcgc cgccagaggg cgccgtagcc caccctttgg aggaactgca ga -             #ggctggcg  16860                                                                  - - cgtgctacgc cggacccggc actcacccgt ggaccgttgc aggtcctgac cg -             #gccttctc  16920                                                                  - - cgcgcagggt cagacggaga ccgcgccact caccacatgg cgctcgaggc tc -             #cgggaacc  16980                                                                  - - gtgcgtggag aaagcctaga cccgcctgtt tcacagaagg ggccagcgcg ca -             #cacgccac  17040                                                                  - - aggccacccc ccgtgcgact gagcttcaac cccgtcaatg ccgatgtacc cg -             #ctacctgg  17100                                                                  - - cgagacgcca ctaacgtgta ctcgggtgct ccctactatg tgtgtgttta cg -             #aacgcggt  17160                                                                  - - ggccgtcagg aagacgactg gctgccgata ccactgagct tcccagaaga gc -             #ccgtgccc  17220                                                                  - - ccgccaccgg gcttagtgtt catggacgac ttgttcatta acacgaagca gt -             #gcgacttt  17280                                                                  - - gtggacacgc tagaggccgc ctgtcgcacg caaggctaca cgttgagaca gc -             #gcgtgcct  17340                                                                  - - gtcgccattc ctcgcgacgc ggaaatcgca gacgcagtta aatcgcactt tt -             #tagaggcg  17400                                                                  - - tgcctagtgt tacgggggct ggcttcggag gctagtgcct ggataagagc tg -             #ccacgtcc  17460                                                                  - - ccgccccttg gccgccacgc ctgctggatg gacgtgttag gattatggga aa -             #gccgcccc  17520                                                                  - - cacactctag gtttggagtt acgcggcgta aactgtggcg gcacggacgg tg -             #actggtta  17580                                                                  - - gagattttaa aacagcccga tgtgcaaaag acagtcagcg ggagtcttgt gg -             #catgcgtg  17640                                                                  - - atcgtcacac ccgcattgga agcctggctt gtgttacctg ggggttttgc ta -             #ttaaagcc  17700                                                                  - - cgctataggg cgtcgaagga ggatctggtg ttcattcgag gccgctatgg ct -             #agccggag  17760                                                                  - - gcgcaaactt cggaatttcc taaacaagga atgcatatgg actgttaacc ca -             #atgtcagg  17820                                                                  - - ggaccatatc aaggtcttta acgcctgcac ctctatctcg ccggtgtatg ac -             #cctgagct  17880                                                                  - - ggtaaccagc tacgcactga gcgtgcctgc ttacaatgtg tctgtggcta tc -             #ttgctgca  17940                                                                  - - taaagtcatg ggaccgtgtg tggctgtggg aattaacgga gaaatgatca tg -             #tacgtcgt  18000                                                                  - - aagccagtgt gtttctgtgc ggcccgtccc ggggcgcgat ggtatggcgc tc -             #atctactt  18060                                                                  - - tggacagttt ctggaggaag catccggact gagatttccc tacattgctc cg -             #ccgccgtc  18120                                                                  - - gcgcgaacac gtacctgacc tgaccagaca agaattagtt catacctccc ag -             #gtggtgcg  18180                                                                  - - ccgcggcgac ctgaccaatt gcactatggg tctcgaattc aggaatgtga ac -             #ccttttgt  18240                                                                  - - ttggctcggg ggcggatcgg tgtggctgct gttcttgggc gtggactaca tg -             #gcgttctg  18300                                                                  - - tccgggtgtc gacggaatgc cgtcgttggc aagagtggcc gccctgctta cc -             #aggtgcga  18360                                                                  - - ccacccagac tgtgtccact gccatggact ccgtggacac gttaatgtat tt -             #cgtgggta  18420                                                                  - - ctgttctgcg cagtcgccgg gtctatctaa catctgtccc tgtatcaaat ca -             #tgtgggac  18480                                                                  - - cgggaatgga gtgactaggg tcactggaaa cagaaatttt ctgggtcttc tg -             #ttcgatcc  18540                                                                  - - cattgtccag agcagggtaa cagctctgaa gataactagc cacccaaccc cc -             #acgcacgt  18600                                                                  - - cgagaatgtg ctaacaggag tgctcgacga cggcaccttg gtgccgtccg tc -             #caaggcac  18660                                                                  - - cctgggtcct cttacgaatg tctgactact tcagccgctt gctgatatat ga -             #gtgtaaaa  18720                                                                  - - aacttaaggc cctgggctta cgttcttatt gaagcatgtt gcgcacatca gc -             #gagctgga  18780                                                                  - - ccgtcctccg ggtcgcgtgt agattatggt tccgttctcc ttcttgatgt tt -             #aaattttt  18840                                                                  - - gggggggaac caccgacaaa gcgtctttat gatttccgcg aacacggagt tg -             #gctacgtg  18900                                                                  - - cttttggtgg gctacgtacc caatgttaat gttctctacg gatgccagta gc -             #atgctgat  18960                                                                  - - gatcgccacc actatccatg tctttccgtg tctccttggt attaggaata cg -             #cttgcctt  19020                                                                  - - ttgcttaaac gtctgtaaaa cactgtttgg agtttcaaat aaaccgaagt ac -             #tgcttaaa  19080                                                                  - - caatccaaac aactggtgcg tcttttgtgg ggccttgatt gaaaccaaaa ag -             #aaaaaagt  19140                                                                  - - gtgcattact agctgctgtt ggaagggctc cagccagtgc accccgggaa cg -             #taacagcc  19200                                                                  - - gttcagaaag gacgaaaggt taaccagaaa agcctgaagt tcgcggtaga ca -             #gagcaggc  19260                                                                  - - gtgcagggag tcgtgtgttt ttctgcccgc ctggtactcg accagttgat cg -             #gccgtgga  19320                                                                  - - gacgtgcgcg tcctcgcgca cacaccgcat ctgcaagtat gttgataggg ac -             #tccaatag  19380                                                                  - - gcgcggcttt gcggggacgt tgtcctcgga cggtctgggg gttcccacgt cg -             #ggatttgc  19440                                                                  - - tgacgtgggc gtggcgggat ggtgccgtgt gcagtatgtt tccaggaccg aa -             #ctgtatga  19500                                                                  - - gtttattctg tgcaccacgc caataaaagg gtgcgccatc cgtgccgttt tg -             #ggacagtg  19560                                                                  - - tcgcgtgaat gtcggggcac tcagttccca cctctctccg gcgtctttgg cg -             #gtctcctc  19620                                                                  - - caggttggcg gcaaggcgct ccctgtgacg gctgagcagc atgtttgctt tg -             #agctcgct  19680                                                                  - - cgtgtccgag ggtgacccgg aggtgaccag taggtacgtc aagggcgtac aa -             #cttgccct  19740                                                                  - - ggaccttagc gagaacacac ctggacaatt taagttgata gaaactcccc tg -             #aacagctt  19800                                                                  - - cctcttggtt tccaacgtga tgcccgaggt ccagccaatc tgcagtggcc gg -             #ccggcctt  19860                                                                  - - gcggccagac tttagtaatc tccacttgcc tagactggag aagctccaga ga -             #gtcctcgg  19920                                                                  - - gcagggtttc ggggcggcgg gtgaggaaat cgcactggac ccgtctcacg ta -             #gaaacaca  19980                                                                  - - cgaaaagggc caggtgttct acaaccacta tgctaccgag gagtggacgt gg -             #gctttgac  20040                                                                  - - tctgaataag gatgcgctcc ttcgggaggc tgtagatggc ctgtgtgacc cc -             #ggaacttg  20100                                                                  - - gaagggtctt cttcctgacg acccccttcc gttgctatgg ctgctgttca ac -             #ggacccgc  20160                                                                  - - ctctttttgt cgggccgact gttgcctgta caagcagcac tgcggttacc cg -             #ggcccggt  20220                                                                  - - gctacttcca ggtcacatgt acgctcccaa acgggatctt ttgtcgttcg tt -             #aatcatgc  20280                                                                  - - cctgaagtac accaagtttc tatacggaga tttttccggg acatgggcgg cg -             #gcttgccg  20340                                                                  - - cccgccattc gctacttctc ggatacaaag ggtagtgagt cagatgaaaa tc -             #atagatgc  20400                                                                  - - ttccgacact tacatttccc acacctgcct cttgtgtcac atatatcagc aa -             #aatagcat  20460                                                                  - - aattgcgggt caggggaccc acgtgggtgg aatcctactg ttgagtggaa aa -             #gggaccca  20520                                                                  - - gtatataaca ggcaatgttc agacccaaag gtgtccaact acgggcgact at -             #ctaatcat  20580                                                                  - - cccatcgtat gacataccgg cgatcatcac catgatcaag gagaatggac tc -             #aaccaact  20640                                                                  - - ctaaaagaga gtttattaag tcggctctgg aggccaacat caacaggagg gc -             #agctgtat  20700                                                                  - - cgctatttga                - #                  - #                       - #     20710                                                                   - -  - - <210> SEQ ID NO 2                                                    <211> LENGTH: 4128                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 2                                                          - - atggaggcga ccttggagca acgacctttc ccgtacctcg ccacggaggc ca -              #acctccta     60                                                                  - - acgcagatta aggagtcggc tgccgacgga ctcttcaaga gctttcagct at -             #tgctcggc    120                                                                  - - aaggacgcca gagaaggcag tgtccgtttc gaagcgctac tgggcgtata ta -             #ccaatgtg    180                                                                  - - gtggagtttg ttaagtttct ggagaccgcc ctcgccgccg cttgcgtcaa ta -             #ccgagttc    240                                                                  - - aaggacctgc ggagaatgat agatggaaaa atacagttta aaatttcaat gc -             #ccactatt    300                                                                  - - gcccacggag acgggaggag gcccaacaag cagagacagt atatcgtcat ga -             #aggcttgc    360                                                                  - - aataagcacc acatcggtgc ggagattgag cttgcggccg cagacatcga gc -             #ttctcttc    420                                                                  - - gccgagaaag agacgccctt ggacttcaca gagtacgcgg gtgccatcaa ga -             #cgattacg    480                                                                  - - tcggctttgc agtttggtat ggacgcccta gaacgggggc tagtggacac gg -             #ttctcgca    540                                                                  - - gttaaacttc ggcacgctcc acccgtcttt attttaaaga cgctgggcga tc -             #ccgtctac    600                                                                  - - tctgagaggg gcctcaaaaa ggccgtcaag tctgacatgg tatccatgtt ca -             #aggcacac    660                                                                  - - ctcatagaac attcattttt tctagataag gccgagctca tgacaagggg ga -             #agcagtat    720                                                                  - - gtcctaacca tgctctccga catgctggcc gcggtgtgcg aggataccgt ct -             #ttaagggt    780                                                                  - - gtcagcacgt acaccacggc ctctgggcag caggtggccg gcgtcctgga ga -             #cgacggac    840                                                                  - - agcgtcatga gacggctgat gaacctgctg gggcaagtgg aaagtgccat gt -             #ccgggccc    900                                                                  - - gcggcctacg ccagctacgt tgtcaggggt gccaacctcg tcaccgccgt ta -             #gctacgga    960                                                                  - - agggcgatga gaaactttga acagtttatg gcacgcatag tggaccatcc ca -             #acgctctg   1020                                                                  - - ccgtctgtgg aaggtgacaa ggccgctctg gcggacggac acgacgagat tc -             #agagaacc   1080                                                                  - - cgcatcgccg cctctctcgt caagataggg gataagtttg tggccattga aa -             #gtttgcag   1140                                                                  - - cgcatgtaca acgagactca gtttccctgc ccactgaacc ggcgcatcca gt -             #acacctat   1200                                                                  - - ttcttccctg ttggccttca ccttcccgtg ccccgctact cgacatccgt ct -             #cagtcagg   1260                                                                  - - ggcgtagaat ccccggccat ccagtcgacc gagacgtggg tggttaataa aa -             #acaacgtg   1320                                                                  - - cctctttgct tcggttacca aaacgccctc aaaagcatat gccaccctcg aa -             #tgcacaac   1380                                                                  - - cccacccagt cagcccaggc actaaaccaa gcttttcccg atcccgacgg gg -             #gacatggg   1440                                                                  - - tacggtctca ggtatgagca gacgccaaac atgaacctat tcagaacgtt cc -             #accagtat   1500                                                                  - - tacatgggga aaaacgtggc atttgttccc gatgtggccc aaaaagcgct cg -             #taaccacg   1560                                                                  - - gaggatctac tgcacccaac ctctcaccgt ctcctcagat tggaggtcca cc -             #ccttcttt   1620                                                                  - - gatttttttg tgcacccctg tcctggagcg agaggatcgt accgcgccac cc -             #acagaaca   1680                                                                  - - atggttggaa atataccaca accgctcgct ccaagggagt ttcaggaaag ta -             #gaggggcg   1740                                                                  - - cagttcgacg ctgtgacgaa tatgacacac gtcatagacc agctaactat tg -             #acgtcata   1800                                                                  - - caggagacgg catttgaccc cgcgtatccc ctgttctgct atgtaatcga ag -             #caatgatt   1860                                                                  - - cacggacagg aagaaaaatt cgtgatgaac atgcccctca ttgccctggt ca -             #ttcaaacc   1920                                                                  - - tactgggtca actcgggaaa actggcgttt gtgaacagtt atcacatggt ta -             #gattcatc   1980                                                                  - - tgtacgcata ttgggaatgg aagcatccct aaggaggcgc acggccacta cc -             #ggaaaatc   2040                                                                  - - ttaggcgagc tcatcgccct tgagcaggcg cttctcaagc tcgcgggaca cg -             #agacggtg   2100                                                                  - - ggtcggacgc cgatcacaca tctggtttcg gctctcctcg acccgcatct gc -             #tgcctccc   2160                                                                  - - tttgcctacc acgatgtctt tacggatctt atgcagaagt catccagaca ac -             #ccataatc   2220                                                                  - - aagatcgggg atcaaaacta cgacaaccct caaaataggg cgacattcat ca -             #acctcagg   2280                                                                  - - ggtcgcatgg aggacctagt caataacctt gttaacattt accagacaag gg -             #tcaatgag   2340                                                                  - - gaccatgacg agagacacgt cctggacgtg gcgcccctgg acgagaatga ct -             #acaacccg   2400                                                                  - - gtcctcgaga agctattcta ctatgtttta atgccggtgt gcagtaacgg cc -             #acatgtgc   2460                                                                  - - ggtatggggg tcgactatca aaacgtggcc ctgacgctga cttacaacgg cc -             #ccgtcttt   2520                                                                  - - gcggacgtcg tgaacgcaca ggatgatatt ctactgcacc tggagaacgg aa -             #ccttgaag   2580                                                                  - - gacattctgc aggcaggcga catacgcccg acggtggaca tgatcagggt gc -             #tgtgcacc   2640                                                                  - - tcgtttctga cgtgcccttt cgtcacccag gccgctcgcg tgatcacaaa gc -             #gggacccg   2700                                                                  - - gcccagagtt ttgccacgca cgaatacggg aaggatgtgg cgcagaccgt gc -             #ttgttaat   2760                                                                  - - ggctttggtg cgttcgcggt ggcggaccgc tctcgcgagg cggcggagac ta -             #tgttttat   2820                                                                  - - ccggtaccct ttaacaagct ctacgctgac ccgttggtgg ctgccacact gc -             #atccgctc   2880                                                                  - - ctgccaaact atgtcaccag gctccccaac cagagaaacg cggtggtctt ta -             #acgtgcca   2940                                                                  - - tccaatctca tggcagaata tgaggaatgg cacaagtcgc ccgtcgcggc gt -             #atgccgcg   3000                                                                  - - tcttgtcagg ccaccccggg cgccattagc gccatggtga gcatgcacca aa -             #aactatct   3060                                                                  - - gcccccagtt tcatttgcca ggcaaaacac cgcatgcacc ctggttttgc ca -             #tgacagtc   3120                                                                  - - gtcaggacgg acgaggttct agcagagcac atcctatact gctccagggc gt -             #cgacatcc   3180                                                                  - - atgtttgtgg gcttgccttc ggtggtacgg cgcgaggtac gttcggacgc gg -             #tgactttt   3240                                                                  - - gaaattaccc acgagatcgc ttccctgcac accgcacttg gctactcatc ag -             #tcatcgcc   3300                                                                  - - ccggcccacg tggccgccat aactacagac atgggagtac attgtcagga cc -             #tctttatg   3360                                                                  - - attttcccag gggacgcgta tcaggaccgc cagctgcatg actatatcaa aa -             #tgaaagcg   3420                                                                  - - ggcgtgcaaa ccggctcacc gggaaacaga atggatcacg tgggatacac tg -             #ctggggtt   3480                                                                  - - cctcgctgcg agaacctgcc cggtttgagt catggtcagc tggcaacctg cg -             #agataatt   3540                                                                  - - cccacgccgg tcacatctga cgttgcctat ttccagaccc ccagcaaccc cc -             #gggggcgt   3600                                                                  - - gcggcgtcgg tcgtgtcgtg tgatgcttac agtaacgaaa gcgcagagcg tt -             #tgttctac   3660                                                                  - - gaccattcaa taccagaccc cgcgtacgaa tgccggtcca ccaacaaccc gt -             #gggcttcg   3720                                                                  - - cagcgtggct ccctcggcga cgtgctatac aatatcacct ttcgccagac tg -             #cgctgccg   3780                                                                  - - ggcatgtaca gtccttgtcg gcagttcttc cacaaggaag acattatgcg gt -             #acaatagg   3840                                                                  - - gggttgtaca ctttggttaa tgagtattct gccaggcttg ctggggcccc cg -             #ccaccagc   3900                                                                  - - actacagacc tccagtacgt cgtggtcaac ggtacagacg tgtttttgga cc -             #agccttgc   3960                                                                  - - catatgctgc aggaggccta tcccacgctc gccgccagcc acagagttat gc -             #ttgccgag   4020                                                                  - - tacatgtcaa acaagcagac acacgcccca gtacacatgg gccagtatct ca -             #ttgaagag   4080                                                                  - - gtggcgccga tgaagagact attaaagctc ggaaacaagg tggtgtat  - #                   4128                                                                         - -  - - <210> SEQ ID NO 3                                                    <211> LENGTH: 1376                                                             <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 3                                                          - - Met Glu Ala Thr Leu Glu Gln Arg Pro Phe Pr - #o Tyr Leu Ala Thr Glu         1               5 - #                 10 - #                 15               - - Ala Asn Leu Leu Thr Gln Ile Lys Glu Ser Al - #a Ala Asp Gly Leu Phe                    20     - #             25     - #             30                   - - Lys Ser Phe Gln Leu Leu Leu Gly Lys Asp Al - #a Arg Glu Gly Ser Val                35         - #         40         - #         45                       - - Arg Phe Glu Ala Leu Leu Gly Val Tyr Thr As - #n Val Val Glu Phe Val            50             - #     55             - #     60                           - - Lys Phe Leu Glu Thr Ala Leu Ala Ala Ala Cy - #s Val Asn Thr Glu Phe        65                 - # 70                 - # 75                 - # 80        - - Lys Asp Leu Arg Arg Met Ile Asp Gly Lys Il - #e Gln Phe Lys Ile Ser                        85 - #                 90 - #                 95               - - Met Pro Thr Ile Ala His Gly Asp Gly Arg Ar - #g Pro Asn Lys Gln Arg                   100      - #           105      - #           110                   - - Gln Tyr Ile Val Met Lys Ala Cys Asn Lys Hi - #s His Ile Gly Ala Glu               115          - #       120          - #       125                       - - Ile Glu Leu Ala Ala Ala Asp Ile Glu Leu Le - #u Phe Ala Glu Lys Glu           130              - #   135              - #   140                           - - Thr Pro Leu Asp Phe Thr Glu Tyr Ala Gly Al - #a Ile Lys Thr Ile Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Ala Leu Gln Phe Gly Met Asp Ala Leu Gl - #u Arg Gly Leu Val         Asp                                                                                              165  - #               170  - #               175              - - Thr Val Leu Ala Val Lys Leu Arg His Ala Pr - #o Pro Val Phe Ile Leu                   180      - #           185      - #           190                   - - Lys Thr Leu Gly Asp Pro Val Tyr Ser Glu Ar - #g Gly Leu Lys Lys Ala               195          - #       200          - #       205                       - - Val Lys Ser Asp Met Val Ser Met Phe Lys Al - #a His Leu Ile Glu His           210              - #   215              - #   220                           - - Ser Phe Phe Leu Asp Lys Ala Glu Leu Met Th - #r Arg Gly Lys Gln Tyr       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Leu Thr Met Leu Ser Asp Met Leu Ala Al - #a Val Cys Glu Asp         Thr                                                                                              245  - #               250  - #               255              - - Val Phe Lys Gly Val Ser Thr Tyr Thr Thr Al - #a Ser Gly Gln Gln Val                   260      - #           265      - #           270                   - - Ala Gly Val Leu Glu Thr Thr Asp Ser Val Me - #t Arg Arg Leu Met Asn               275          - #       280          - #       285                       - - Leu Leu Gly Gln Val Glu Ser Ala Met Ser Gl - #y Pro Ala Ala Tyr Ala           290              - #   295              - #   300                           - - Ser Tyr Val Val Arg Gly Ala Asn Leu Val Th - #r Ala Val Ser Tyr Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Arg Ala Met Arg Asn Phe Glu Gln Phe Met Al - #a Arg Ile Val Asp         His                                                                                              325  - #               330  - #               335              - - Pro Asn Ala Leu Pro Ser Val Glu Gly Asp Ly - #s Ala Ala Leu Ala Asp                   340      - #           345      - #           350                   - - Gly His Asp Glu Ile Gln Arg Thr Arg Ile Al - #a Ala Ser Leu Val Lys               355          - #       360          - #       365                       - - Ile Gly Asp Lys Phe Val Ala Ile Glu Ser Le - #u Gln Arg Met Tyr Asn           370              - #   375              - #   380                           - - Glu Thr Gln Phe Pro Cys Pro Leu Asn Arg Ar - #g Ile Gln Tyr Thr Tyr       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Phe Phe Pro Val Gly Leu His Leu Pro Val Pr - #o Arg Tyr Ser Thr         Ser                                                                                              405  - #               410  - #               415              - - Val Ser Val Arg Gly Val Glu Ser Pro Ala Il - #e Gln Ser Thr Glu Thr                   420      - #           425      - #           430                   - - Trp Val Val Asn Lys Asn Asn Val Pro Leu Cy - #s Phe Gly Tyr Gln Asn               435          - #       440          - #       445                       - - Ala Leu Lys Ser Ile Cys His Pro Arg Met Hi - #s Asn Pro Thr Gln Ser           450              - #   455              - #   460                           - - Ala Gln Ala Leu Asn Gln Ala Phe Pro Asp Pr - #o Asp Gly Gly His Gly       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Tyr Gly Leu Arg Tyr Glu Gln Thr Pro Asn Me - #t Asn Leu Phe Arg         Thr                                                                                              485  - #               490  - #               495              - - Phe His Gln Tyr Tyr Met Gly Lys Asn Val Al - #a Phe Val Pro Asp Val                   500      - #           505      - #           510                   - - Ala Gln Lys Ala Leu Val Thr Thr Glu Asp Le - #u Leu His Pro Thr Ser               515          - #       520          - #       525                       - - His Arg Leu Leu Arg Leu Glu Val His Pro Ph - #e Phe Asp Phe Phe Val           530              - #   535              - #   540                           - - His Pro Cys Pro Gly Ala Arg Gly Ser Tyr Ar - #g Ala Thr His Arg Thr       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Met Val Gly Asn Ile Pro Gln Pro Leu Ala Pr - #o Arg Glu Phe Gln         Glu                                                                                              565  - #               570  - #               575              - - Ser Arg Gly Ala Gln Phe Asp Ala Val Thr As - #n Met Thr His Val Ile                   580      - #           585      - #           590                   - - Asp Gln Leu Thr Ile Asp Val Ile Gln Glu Th - #r Ala Phe Asp Pro Ala               595          - #       600          - #       605                       - - Tyr Pro Leu Phe Cys Tyr Val Ile Glu Ala Me - #t Ile His Gly Gln Glu           610              - #   615              - #   620                           - - Glu Lys Phe Val Met Asn Met Pro Leu Ile Al - #a Leu Val Ile Gln Thr       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Tyr Trp Val Asn Ser Gly Lys Leu Ala Phe Va - #l Asn Ser Tyr His         Met                                                                                              645  - #               650  - #               655              - - Val Arg Phe Ile Cys Thr His Ile Gly Asn Gl - #y Ser Ile Pro Lys Glu                   660      - #           665      - #           670                   - - Ala His Gly His Tyr Arg Lys Ile Leu Gly Gl - #u Leu Ile Ala Leu Glu               675          - #       680          - #       685                       - - Gln Ala Leu Leu Lys Leu Ala Gly His Glu Th - #r Val Gly Arg Thr Pro           690              - #   695              - #   700                           - - Ile Thr His Leu Val Ser Ala Leu Leu Asp Pr - #o His Leu Leu Pro Pro       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Phe Ala Tyr His Asp Val Phe Thr Asp Leu Me - #t Gln Lys Ser Ser         Arg                                                                                              725  - #               730  - #               735              - - Gln Pro Ile Ile Lys Ile Gly Asp Gln Asn Ty - #r Asp Asn Pro Gln Asn                   740      - #           745      - #           750                   - - Arg Ala Thr Phe Ile Asn Leu Arg Gly Arg Me - #t Glu Asp Leu Val Asn               755          - #       760          - #       765                       - - Asn Leu Val Asn Ile Tyr Gln Thr Arg Val As - #n Glu Asp His Asp Glu           770              - #   775              - #   780                           - - Arg His Val Leu Asp Val Ala Pro Leu Asp Gl - #u Asn Asp Tyr Asn Pro       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Val Leu Glu Lys Leu Phe Tyr Tyr Val Leu Me - #t Pro Val Cys Ser         Asn                                                                                              805  - #               810  - #               815              - - Gly His Met Cys Gly Met Gly Val Asp Tyr Gl - #n Asn Val Ala Leu Thr                   820      - #           825      - #           830                   - - Leu Thr Tyr Asn Gly Pro Val Phe Ala Asp Va - #l Val Asn Ala Gln Asp               835          - #       840          - #       845                       - - Asp Ile Leu Leu His Leu Glu Asn Gly Thr Le - #u Lys Asp Ile Leu Gln           850              - #   855              - #   860                           - - Ala Gly Asp Ile Arg Pro Thr Val Asp Met Il - #e Arg Val Leu Cys Thr       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Ser Phe Leu Thr Cys Pro Phe Val Thr Gln Al - #a Ala Arg Val Ile         Thr                                                                                              885  - #               890  - #               895              - - Lys Arg Asp Pro Ala Gln Ser Phe Ala Thr Hi - #s Glu Tyr Gly Lys Asp                   900      - #           905      - #           910                   - - Val Ala Gln Thr Val Leu Val Asn Gly Phe Gl - #y Ala Phe Ala Val Ala               915          - #       920          - #       925                       - - Asp Arg Ser Arg Glu Ala Ala Glu Thr Met Ph - #e Tyr Pro Val Pro Phe           930              - #   935              - #   940                           - - Asn Lys Leu Tyr Ala Asp Pro Leu Val Ala Al - #a Thr Leu His Pro Leu       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Leu Pro Asn Tyr Val Thr Arg Leu Pro Asn Gl - #n Arg Asn Ala Val         Val                                                                                              965  - #               970  - #               975              - - Phe Asn Val Pro Ser Asn Leu Met Ala Glu Ty - #r Glu Glu Trp His Lys                   980      - #           985      - #           990                   - - Ser Pro Val Ala Ala Tyr Ala Ala Ser Cys Gl - #n Ala Thr Pro Gly Ala               995          - #      1000           - #     1005                       - - Ile Ser Ala Met Val Ser Met His Gln Lys Le - #u Ser Ala Pro Ser Phe          1010              - #  1015               - # 1020                           - - Ile Cys Gln Ala Lys His Arg Met His Pro Gl - #y Phe Ala Met Thr Val       1025               1030 - #               1035  - #              1040           - - Val Arg Thr Asp Glu Val Leu Ala Glu His Il - #e Leu Tyr Cys Ser Arg                      1045  - #              1050   - #             1055               - - Ala Ser Thr Ser Met Phe Val Gly Leu Pro Se - #r Val Val Arg Arg Glu                  1060      - #          1065       - #         1070                   - - Val Arg Ser Asp Ala Val Thr Phe Glu Ile Th - #r His Glu Ile Ala Ser              1075          - #      1080           - #     1085                       - - Leu His Thr Ala Leu Gly Tyr Ser Ser Val Il - #e Ala Pro Ala His Val          1090              - #  1095               - # 1100                           - - Ala Ala Ile Thr Thr Asp Met Gly Val His Cy - #s Gln Asp Leu Phe Met       1105               1110 - #               1115  - #              1120           - - Ile Phe Pro Gly Asp Ala Tyr Gln Asp Arg Gl - #n Leu His Asp Tyr Ile                      1125  - #              1130   - #             1135               - - Lys Met Lys Ala Gly Val Gln Thr Gly Ser Pr - #o Gly Asn Arg Met Asp                  1140      - #          1145       - #         1150                   - - His Val Gly Tyr Thr Ala Gly Val Pro Arg Cy - #s Glu Asn Leu Pro Gly              1155          - #      1160           - #     1165                       - - Leu Ser His Gly Gln Leu Ala Thr Cys Glu Il - #e Ile Pro Thr Pro Val          1170              - #  1175               - # 1180                           - - Thr Ser Asp Val Ala Tyr Phe Gln Thr Pro Se - #r Asn Pro Arg Gly Arg       1185               1190 - #               1195  - #              1200           - - Ala Ala Ser Val Val Ser Cys Asp Ala Tyr Se - #r Asn Glu Ser Ala Glu                      1205  - #              1210   - #             1215               - - Arg Leu Phe Tyr Asp His Ser Ile Pro Asp Pr - #o Ala Tyr Glu Cys Arg                  1220      - #          1225       - #         1230                   - - Ser Thr Asn Asn Pro Trp Ala Ser Gln Arg Gl - #y Ser Leu Gly Asp Val              1235          - #      1240           - #     1245                       - - Leu Tyr Asn Ile Thr Phe Arg Gln Thr Ala Le - #u Pro Gly Met Tyr Ser          1250              - #  1255               - # 1260                           - - Pro Cys Arg Gln Phe Phe His Lys Glu Asp Il - #e Met Arg Tyr Asn Arg       1265               1270 - #               1275  - #              1280           - - Gly Leu Tyr Thr Leu Val Asn Glu Tyr Ser Al - #a Arg Leu Ala Gly Ala                      1285  - #              1290   - #             1295               - - Pro Ala Thr Ser Thr Thr Asp Leu Gln Tyr Va - #l Val Val Asn Gly Thr                  1300      - #          1305       - #         1310                   - - Asp Val Phe Leu Asp Gln Pro Cys His Met Le - #u Gln Glu Ala Tyr Pro              1315          - #      1320           - #     1325                       - - Thr Leu Ala Ala Ser His Arg Val Met Leu Al - #a Glu Tyr Met Ser Asn          1330              - #  1335               - # 1340                           - - Lys Gln Thr His Ala Pro Val His Met Gly Gl - #n Tyr Leu Ile Glu Glu       1345               1350 - #               1355  - #              1360           - - Val Ala Pro Met Lys Arg Leu Leu Lys Leu Gl - #y Asn Lys Val Val Tyr                      1365  - #              1370   - #             1375               - -  - - <210> SEQ ID NO 4                                                    <211> LENGTH: 1143                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 4                                                          - - agcattcggg gacagacctt taacctgctc tacgtagacg aggcgaattt ta -              #ttaaaaag     60                                                                  - - gatgcactgc cggctattct gggtttcatg cttcagaaag acgccaagct ta -             #tatttata    120                                                                  - - tcatccgtga actcgtcaga ccgctccacg agtttcctgc ttaacctcag ga -             #acgcccag    180                                                                  - - gaaaagatgc tgaatgtggt cagttacgtg tgtgcggacc accgagaaga tt -             #tccacctg    240                                                                  - - caagacgcac tagtgtcctg tccttgttac agactgcaca ttccgacgta ca -             #tcaccatc    300                                                                  - - gacgaatcca tcaaaaccac caccaacctc tttatggagg gggcattcga ca -             #ccgaacta    360                                                                  - - atgggcgagg gagcagcgtc gtcaaatgct acgctttacc gcgtggtggg tg -             #acgcagcg    420                                                                  - - ctgacacagt ttgacatgtg tcgggtagac accaccgccc aggaggttca ga -             #agtgcctt    480                                                                  - - ggaaaacagc tgtttgttta catcgacccc gcgtatacga acaacacgga gg -             #cgtccggt    540                                                                  - - actggcgtgg gcgccgttgt cacgagtact cagactccca ccagaagcct ca -             #tattgggc    600                                                                  - - atggagcatt tcttcctgcg cgacctcact ggcgcagctg cttacgagat ag -             #cgtcctgc    660                                                                  - - gcatgcacga tgattaaggc gatcgctgtg ctccacacca caattgagcg cg -             #tgaacgcg    720                                                                  - - gcggtcgaag gcaacagcag ccaagattct ggggtggcca ttgcaaccgt cc -             #ttaacgaa    780                                                                  - - atatgcccgc tccccataca ttttctacac tatactgaca agagcagcgc cc -             #tgcagtgg    840                                                                  - - ccaatttaca tgttgggagg cgagaaatcc tccgcgtttg agacattcat ct -             #acgctctg    900                                                                  - - aactccggca ccctgagcgc cagccagacg gtggtgtcca acaccatcaa aa -             #tatcattt    960                                                                  - - gacccggtga cctacctggt agaacaggtc cgcgcgatca agtgcgtccc gc -             #ttagggat   1020                                                                  - - ggagggcagt catacagcgc caagcaaaag cacatgtcgg acgacttact tg -             #tggcagtt   1080                                                                  - - gtcatggccc attttatggc taccgatgat agacacatgt acaagcccat at -             #ccccacaa   1140                                                                  - - taa                  - #                  - #                  - #                1143                                                                   - -  - - <210> SEQ ID NO 5                                                    <211> LENGTH: 380                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 5                                                          - - Ser Ile Arg Gly Gln Thr Phe Asn Leu Leu Ty - #r Val Asp Glu Ala Asn         1               5 - #                 10 - #                 15               - - Phe Ile Lys Lys Asp Ala Leu Pro Ala Ile Le - #u Gly Phe Met Leu Gln                    20     - #             25     - #             30                   - - Lys Asp Ala Lys Leu Ile Phe Ile Ser Ser Va - #l Asn Ser Ser Asp Arg                35         - #         40         - #         45                       - - Ser Thr Ser Phe Leu Leu Asn Leu Arg Asn Al - #a Gln Glu Lys Met Leu            50             - #     55             - #     60                           - - Asn Val Val Ser Tyr Val Cys Ala Asp His Ar - #g Glu Asp Phe His Leu        65                 - # 70                 - # 75                 - # 80        - - Gln Asp Ala Leu Val Ser Cys Pro Cys Tyr Ar - #g Leu His Ile Pro Thr                        85 - #                 90 - #                 95               - - Tyr Ile Thr Ile Asp Glu Ser Ile Lys Thr Th - #r Thr Asn Leu Phe Met                   100      - #           105      - #           110                   - - Glu Gly Ala Phe Asp Thr Glu Leu Met Gly Gl - #u Gly Ala Ala Ser Ser               115          - #       120          - #       125                       - - Asn Ala Thr Leu Tyr Arg Val Val Gly Asp Al - #a Ala Leu Thr Gln Phe           130              - #   135              - #   140                           - - Asp Met Cys Arg Val Asp Thr Thr Ala Gln Gl - #u Val Gln Lys Cys Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gly Lys Gln Leu Phe Val Tyr Ile Asp Pro Al - #a Tyr Thr Asn Asn         Thr                                                                                              165  - #               170  - #               175              - - Glu Ala Ser Gly Thr Gly Val Gly Ala Val Va - #l Thr Ser Thr Gln Thr                   180      - #           185      - #           190                   - - Pro Thr Arg Ser Leu Ile Leu Gly Met Glu Hi - #s Phe Phe Leu Arg Asp               195          - #       200          - #       205                       - - Leu Thr Gly Ala Ala Ala Tyr Glu Ile Ala Se - #r Cys Ala Cys Thr Met           210              - #   215              - #   220                           - - Ile Lys Ala Ile Ala Val Leu His Thr Thr Il - #e Glu Arg Val Asn Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ala Val Glu Gly Asn Ser Ser Gln Asp Ser Gl - #y Val Ala Ile Ala         Thr                                                                                              245  - #               250  - #               255              - - Val Leu Asn Glu Ile Cys Pro Leu Pro Ile Hi - #s Phe Leu His Tyr Thr                   260      - #           265      - #           270                   - - Asp Lys Ser Ser Ala Leu Gln Trp Pro Ile Ty - #r Met Leu Gly Gly Glu               275          - #       280          - #       285                       - - Lys Ser Ser Ala Phe Glu Thr Phe Ile Tyr Al - #a Leu Asn Ser Gly Thr           290              - #   295              - #   300                           - - Leu Ser Ala Ser Gln Thr Val Val Ser Asn Th - #r Ile Lys Ile Ser Phe       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Asp Pro Val Thr Tyr Leu Val Glu Gln Val Ar - #g Ala Ile Lys Cys         Val                                                                                              325  - #               330  - #               335              - - Pro Leu Arg Asp Gly Gly Gln Ser Tyr Ser Al - #a Lys Gln Lys His Met                   340      - #           345      - #           350                   - - Ser Asp Asp Leu Leu Val Ala Val Val Met Al - #a His Phe Met Ala Thr               355          - #       360          - #       365                       - - Asp Asp Arg His Met Tyr Lys Pro Ile Ser Pr - #o Gln                           370              - #   375              - #   380                           - -  - - <210> SEQ ID NO 6                                                    <211> LENGTH: 234                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 6                                                          - - atgggtgagc cagtggatcc tggacatgtg gtgaatgaga aagattttga gg -              #agtgtgaa     60                                                                  - - caatttttca gtcaacccct tagggagcaa gtggtcgcgg gggtcagggc ac -             #tcgacggc    120                                                                  - - ctcggtctcg ctgactctct atgtcacaaa acagaaagac tctgcctgct ga -             #tggacctg    180                                                                  - - gtgggcacgg agtgctttgc gagggtgtgc cgcctagaca ccggtgcgaa at - #ga               234                                                                        - -  - - <210> SEQ ID NO 7                                                    <211> LENGTH: 77                                                               <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 7                                                          - - Met Gly Glu Pro Val Asp Pro Gly His Val Va - #l Asn Glu Lys Asp Phe         1               5 - #                 10 - #                 15               - - Glu Glu Cys Glu Gln Phe Phe Ser Gln Pro Le - #u Arg Glu Gln Val Val                    20     - #             25     - #             30                   - - Ala Gly Val Arg Ala Leu Asp Gly Leu Gly Le - #u Ala Asp Ser Leu Cys                35         - #         40         - #         45                       - - His Lys Thr Glu Arg Leu Cys Leu Leu Met As - #p Leu Val Gly Thr Glu            50             - #     55             - #     60                           - - Cys Phe Ala Arg Val Cys Arg Leu Asp Thr Gl - #y Ala Lys                    65                 - # 70                 - # 75                               - -  - - <210> SEQ ID NO 8                                                    <211> LENGTH: 585                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 8                                                          - - atgaagagtg tggcgagtcc cttatgtcag ttccacggcg tgttttgcct gt -              #accagtgt     60                                                                  - - cgccagtgcc tggcatacca cgtgtgtgat gggggcgccg aatgcgttct cc -             #tgcatacg    120                                                                  - - ccggagagcg tcatctgcga actaacgggt aactgcatgc tcggcaacat tc -             #aagagggc    180                                                                  - - cagtttttag ggccggtacc gtatcggact ttggataacc aggttgacag gg -             #acgcatat    240                                                                  - - cacgggatgc tagcgtgtct gaaacgggac attgtgcggt atttgcagac at -             #ggccggac    300                                                                  - - accaccgtaa tcgtgcagga aatagccctg ggggacggcg tcaccgacac ca -             #tctcggcc    360                                                                  - - attatagatg aaacattcgg tgagtgtctt cccgtactgg gggaggccca ag -             #gcgggtac    420                                                                  - - gccctggtct gtagcatgta tctgcacgtt atcgtctcca tctattcgac aa -             #aaacggtg    480                                                                  - - tacaacagta tgctatttaa atgcacaaag aataaaaagt acgactgcat tg -             #ccaagcgg    540                                                                  - - gtgcggacaa aatggatgcg catgctatca acgaaagata cgtag   - #                      585                                                                         - -  - - <210> SEQ ID NO 9                                                    <211> LENGTH: 194                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 9                                                          - - Met Lys Ser Val Ala Ser Pro Leu Cys Gln Ph - #e His Gly Val Phe Cys         1               5 - #                 10 - #                 15               - - Leu Tyr Gln Cys Arg Gln Cys Leu Ala Tyr Hi - #s Val Cys Asp Gly Gly                    20     - #             25     - #             30                   - - Ala Glu Cys Val Leu Leu His Thr Pro Glu Se - #r Val Ile Cys Glu Leu                35         - #         40         - #         45                       - - Thr Gly Asn Cys Met Leu Gly Asn Ile Gln Gl - #u Gly Gln Phe Leu Gly            50             - #     55             - #     60                           - - Pro Val Pro Tyr Arg Thr Leu Asp Asn Gln Va - #l Asp Arg Asp Ala Tyr        65                 - # 70                 - # 75                 - # 80        - - His Gly Met Leu Ala Cys Leu Lys Arg Asp Il - #e Val Arg Tyr Leu Gln                        85 - #                 90 - #                 95               - - Thr Trp Pro Asp Thr Thr Val Ile Val Gln Gl - #u Ile Ala Leu Gly Asp                   100      - #           105      - #           110                   - - Gly Val Thr Asp Thr Ile Ser Ala Ile Ile As - #p Glu Thr Phe Gly Glu               115          - #       120          - #       125                       - - Cys Leu Pro Val Leu Gly Glu Ala Gln Gly Gl - #y Tyr Ala Leu Val Cys           130              - #   135              - #   140                           - - Ser Met Tyr Leu His Val Ile Val Ser Ile Ty - #r Ser Thr Lys Thr Val       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Tyr Asn Ser Met Leu Phe Lys Cys Thr Lys As - #n Lys Lys Tyr Asp         Cys                                                                                              165  - #               170  - #               175              - - Ile Ala Lys Arg Val Arg Thr Lys Trp Met Ar - #g Met Leu Ser Thr Lys                   180      - #           185      - #           190                   - - Asp Thr                                                                    - -  - - <210> SEQ ID NO 10                                                   <211> LENGTH: 939                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 10                                                         - - atggctagcc ggaggcgcaa acttcggaat ttcctaaaca aggaatgcat at -              #ggactgtt     60                                                                  - - aacccaatgt caggggacca tatcaaggtc tttaacgcct gcacctctat ct -             #cgccggtg    120                                                                  - - tatgaccctg agctggtaac cagctacgca ctgagcgtgc ctgcttacaa tg -             #tgtctgtg    180                                                                  - - gctatcttgc tgcataaagt catgggaccg tgtgtggctg tgggaattaa cg -             #gagaaatg    240                                                                  - - atcatgtacg tcgtaagcca gtgtgtttct gtgcggcccg tcccggggcg cg -             #atggtatg    300                                                                  - - gcgctcatct actttggaca gtttctggag gaagcatccg gactgagatt tc -             #cctacatt    360                                                                  - - gctccgccgc cgtcgcgcga acacgtacct gacctgacca gacaagaatt ag -             #ttcatacc    420                                                                  - - tcccaggtgg tgcgccgcgg cgacctgacc aattgcacta tgggtctcga at -             #tcaggaat    480                                                                  - - gtgaaccctt ttgtttggct cgggggcgga tcggtgtggc tgctgttctt gg -             #gcgtggac    540                                                                  - - tacatggcgt tctgtccggg tgtcgacgga atgccgtcgt tggcaagagt gg -             #ccgccctg    600                                                                  - - cttaccaggt gcgaccaccc agactgtgtc cactgccatg gactccgtgg ac -             #acgttaat    660                                                                  - - gtatttcgtg ggtactgttc tgcgcagtcg ccgggtctat ctaacatctg tc -             #cctgtatc    720                                                                  - - aaatcatgtg ggaccgggaa tggagtgact agggtcactg gaaacagaaa tt -             #ttctgggt    780                                                                  - - cttctgttcg atcccattgt ccagagcagg gtaacagctc tgaagataac ta -             #gccaccca    840                                                                  - - acccccacgc acgtcgagaa tgtgctaaca ggagtgctcg acgacggcac ct -             #tggtgccg    900                                                                  - - tccgtccaag gcaccctggg tcctcttacg aatgtctga      - #                       - #   939                                                                      - -  - - <210> SEQ ID NO 11                                                   <211> LENGTH: 312                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 11                                                         - - Met Ala Ser Arg Arg Arg Lys Leu Arg Asn Ph - #e Leu Asn Lys Glu Cys         1               5 - #                 10 - #                 15               - - Ile Trp Thr Val Asn Pro Met Ser Gly Asp Hi - #s Ile Lys Val Phe Asn                    20     - #             25     - #             30                   - - Ala Cys Thr Ser Ile Ser Pro Val Tyr Asp Pr - #o Glu Leu Val Thr Ser                35         - #         40         - #         45                       - - Tyr Ala Leu Ser Val Pro Ala Tyr Asn Val Se - #r Val Ala Ile Leu Leu            50             - #     55             - #     60                           - - His Lys Val Met Gly Pro Cys Val Ala Val Gl - #y Ile Asn Gly Glu Met        65                 - # 70                 - # 75                 - # 80        - - Ile Met Tyr Val Val Ser Gln Cys Val Ser Va - #l Arg Pro Val Pro Gly                        85 - #                 90 - #                 95               - - Arg Asp Gly Met Ala Leu Ile Tyr Phe Gly Gl - #n Phe Leu Glu Glu Ala                   100      - #           105      - #           110                   - - Ser Gly Leu Arg Phe Pro Tyr Ile Ala Pro Pr - #o Pro Ser Arg Glu His               115          - #       120          - #       125                       - - Val Pro Asp Leu Thr Arg Gln Glu Leu Val Hi - #s Thr Ser Gln Val Val           130              - #   135              - #   140                           - - Arg Arg Gly Asp Leu Thr Asn Cys Thr Met Gl - #y Leu Glu Phe Arg Asn       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Val Asn Pro Phe Val Trp Leu Gly Gly Gly Se - #r Val Trp Leu Leu         Phe                                                                                              165  - #               170  - #               175              - - Leu Gly Val Asp Tyr Met Ala Phe Cys Pro Gl - #y Val Asp Gly Met Pro                   180      - #           185      - #           190                   - - Ser Leu Ala Arg Val Ala Ala Leu Leu Thr Ar - #g Cys Asp His Pro Asp               195          - #       200          - #       205                       - - Cys Val His Cys His Gly Leu Arg Gly His Va - #l Asn Val Phe Arg Gly           210              - #   215              - #   220                           - - Tyr Cys Ser Ala Gln Ser Pro Gly Leu Ser As - #n Ile Cys Pro Cys Ile       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Lys Ser Cys Gly Thr Gly Asn Gly Val Thr Ar - #g Val Thr Gly Asn         Arg                                                                                              245  - #               250  - #               255              - - Asn Phe Leu Gly Leu Leu Phe Asp Pro Ile Va - #l Gln Ser Arg Val Thr                   260      - #           265      - #           270                   - - Ala Leu Lys Ile Thr Ser His Pro Thr Pro Th - #r His Val Glu Asn Val               275          - #       280          - #       285                       - - Leu Thr Gly Val Leu Asp Asp Gly Thr Leu Va - #l Pro Ser Val Gln Gly           290              - #   295              - #   300                           - - Thr Leu Gly Pro Leu Thr Asn Val                                           305                 3 - #10                                                     - -  - - <210> SEQ ID NO 12                                                   <211> LENGTH: 86                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 12                                                         - - atggactcaa ccaactctaa aagagagttt attaagtcgg ctctggaggc ca -              #acatcaac     60                                                                  - - aggagggcag ctgtatcgct atttga          - #                  - #                   86                                                                      - -  - - <210> SEQ ID NO 13                                                   <211> LENGTH: 28                                                               <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 13                                                         - - Met Asp Ser Thr Asn Ser Lys Arg Glu Phe Il - #e Lys Ser Ala Leu Glu         1               5 - #                 10 - #                 15               - - Ala Asn Ile Asn Arg Arg Ala Ala Val Ser Le - #u Phe                                    20     - #             25                                          - -  - - <210> SEQ ID NO 14                                                   <211> LENGTH: 1743                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 14                                                         - - atggcagaag gcggttttgg agcggactcg gtggggcgcg gcggagaaaa gg -              #cctctgtg     60                                                                  - - actaggggag gcaggtggga cttggggagc tcggacgacg aatcaagcac ct -             #ccacaacc    120                                                                  - - agcacggata tggacgacct ccctgaggag aggaaaccac taacgggaaa gt -             #ctgtaaaa    180                                                                  - - acctcgtaca tatacgacgt gcccaccgtc ccgaccagca agccgtggca tt -             #taatgcac    240                                                                  - - gacaactccc tctacgcaac gcctaggttt ccgcccagac ctctcatacg gc -             #acccttcc    300                                                                  - - gaaaaaggca gcatttttgc cagtcggttg tcagcgactg acgacgactc gg -             #gagactac    360                                                                  - - gcgccaatgg atcgcttcgc cttccagagc cccagggtgt gtggtcgccc tc -             #cccttccg    420                                                                  - - cctccaaatc acccacctcc ggcaactagg ccggcagacg cgtcaatggg gg -             #acgtgggc    480                                                                  - - tgggcggatc tgcagggact caagaggacc ccaaagggat ttttaaaaac at -             #ctaccaag    540                                                                  - - gggggcagtc tcaaagcccg tggacgcgat gtaggtgacc gtctcaggga cg -             #gcggcttt    600                                                                  - - gcctttagtc ctaggggcgt gaaatctgcc atagggcaaa acattaaatc at -             #ggttgggg    660                                                                  - - atcggagaat catcggcgac tgctgtcccc gtcaccacgc agcttatggt ac -             #cggtgcac    720                                                                  - - ctcattagaa cgcctgtgac cgtggactac aggaatgttt atttgcttta ct -             #tagagggg    780                                                                  - - gtaatgggtg tgggcaaatc aacgctggtc aacgccgtgt gcgggatctt gc -             #cccaggag    840                                                                  - - agagtgacaa gttttcccga gcccatggtg tactggacga gggcatttac ag -             #attgttac    900                                                                  - - aaggaaattt cccacctgat gaagtctggt aaggcgggag acccgctgac gt -             #ctgccaaa    960                                                                  - - atatactcat gccaaaacaa gttttcgctc cccttccgga cgaacgccac cg -             #ctatcctg   1020                                                                  - - cgaatgatgc agccctggaa cgttgggggt gggtctggga ggggcactca ct -             #ggtgcgtc   1080                                                                  - - tttgataggc atctcctctc cccagcagtg gtgttccctc tcatgcacct ga -             #agcacggc   1140                                                                  - - cgcctatctt ttgatcactt ctttcaatta ctttccatct ttagagccac ag -             #aaggcgac   1200                                                                  - - gtggtcgcca ttctcaccct ctccagcgcc gagtcgttgc ggcgggtcag gg -             #cgagggga   1260                                                                  - - agaaagaacg acgggacggt ggagcaaaac tacatcagag aattggcgtg gg -             #cttatcac   1320                                                                  - - gccgtgtact gttcatggat catgttgcag tacatcactg tggagcagat gg -             #tacaacta   1380                                                                  - - tgcgtacaaa ccacaaatat tccggaaatc tgcttccgca gcgtgcgcct gg -             #cacacaag   1440                                                                  - - gaggaaactt tgaaaaacct tcacgagcag agcatgctac ctatgatcac cg -             #gtgtactg   1500                                                                  - - gatcccgtga gacatcatcc cgtcgtgatc gagctttgct tttgtttctt ca -             #cagagctg   1560                                                                  - - agaaaattac aatttatcgt agccgacgcg gataagttcc acgacgacgt at -             #gcggcctg   1620                                                                  - - tggaccgaaa tctacaggca gatcctgtcc aatccggcta ttaaacccag gg -             #ccatcaac   1680                                                                  - - tggccagcat tagagagcca gtctaaagca gttaatcacc tagaggagac at -             #gcagggtc   1740                                                                  - - tag                  - #                  - #                  - #                1743                                                                   - -  - - <210> SEQ ID NO 15                                                   <211> LENGTH: 580                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 15                                                         - - Met Ala Glu Gly Gly Phe Gly Ala Asp Ser Va - #l Gly Arg Gly Gly Glu         1               5 - #                 10 - #                 15               - - Lys Ala Ser Val Thr Arg Gly Gly Arg Trp As - #p Leu Gly Ser Ser Asp                    20     - #             25     - #             30                   - - Asp Glu Ser Ser Thr Ser Thr Thr Ser Thr As - #p Met Asp Asp Leu Pro                35         - #         40         - #         45                       - - Glu Glu Arg Lys Pro Leu Thr Gly Lys Ser Va - #l Lys Thr Ser Tyr Ile            50             - #     55             - #     60                           - - Tyr Asp Val Pro Thr Val Pro Thr Ser Lys Pr - #o Trp His Leu Met His        65                 - # 70                 - # 75                 - # 80        - - Asp Asn Ser Leu Tyr Ala Thr Pro Arg Phe Pr - #o Pro Arg Pro Leu Ile                        85 - #                 90 - #                 95               - - Arg His Pro Ser Glu Lys Gly Ser Ile Phe Al - #a Ser Arg Leu Ser Ala                   100      - #           105      - #           110                   - - Thr Asp Asp Asp Ser Gly Asp Tyr Ala Pro Me - #t Asp Arg Phe Ala Phe               115          - #       120          - #       125                       - - Gln Ser Pro Arg Val Cys Gly Arg Pro Pro Le - #u Pro Pro Pro Asn His           130              - #   135              - #   140                           - - Pro Pro Pro Ala Thr Arg Pro Ala Asp Ala Se - #r Met Gly Asp Val Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Trp Ala Asp Leu Gln Gly Leu Lys Arg Thr Pr - #o Lys Gly Phe Leu         Lys                                                                                              165  - #               170  - #               175              - - Thr Ser Thr Lys Gly Gly Ser Leu Lys Ala Ar - #g Gly Arg Asp Val Gly                   180      - #           185      - #           190                   - - Asp Arg Leu Arg Asp Gly Gly Phe Ala Phe Se - #r Pro Arg Gly Val Lys               195          - #       200          - #       205                       - - Ser Ala Ile Gly Gln Asn Ile Lys Ser Trp Le - #u Gly Ile Gly Glu Ser           210              - #   215              - #   220                           - - Ser Ala Thr Ala Val Pro Val Thr Thr Gln Le - #u Met Val Pro Val His       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Ile Arg Thr Pro Val Thr Val Asp Tyr Ar - #g Asn Val Tyr Leu         Leu                                                                                              245  - #               250  - #               255              - - Tyr Leu Glu Gly Val Met Gly Val Gly Lys Se - #r Thr Leu Val Asn Ala                   260      - #           265      - #           270                   - - Val Cys Gly Ile Leu Pro Gln Glu Arg Val Th - #r Ser Phe Pro Glu Pro               275          - #       280          - #       285                       - - Met Val Tyr Trp Thr Arg Ala Phe Thr Asp Cy - #s Tyr Lys Glu Ile Ser           290              - #   295              - #   300                           - - His Leu Met Lys Ser Gly Lys Ala Gly Asp Pr - #o Leu Thr Ser Ala Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ile Tyr Ser Cys Gln Asn Lys Phe Ser Leu Pr - #o Phe Arg Thr Asn         Ala                                                                                              325  - #               330  - #               335              - - Thr Ala Ile Leu Arg Met Met Gln Pro Trp As - #n Val Gly Gly Gly Ser                   340      - #           345      - #           350                   - - Gly Arg Gly Thr His Trp Cys Val Phe Asp Ar - #g His Leu Leu Ser Pro               355          - #       360          - #       365                       - - Ala Val Val Phe Pro Leu Met His Leu Lys Hi - #s Gly Arg Leu Ser Phe           370              - #   375              - #   380                           - - Asp His Phe Phe Gln Leu Leu Ser Ile Phe Ar - #g Ala Thr Glu Gly Asp       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Val Val Ala Ile Leu Thr Leu Ser Ser Ala Gl - #u Ser Leu Arg Arg         Val                                                                                              405  - #               410  - #               415              - - Arg Ala Arg Gly Arg Lys Asn Asp Gly Thr Va - #l Glu Gln Asn Tyr Ile                   420      - #           425      - #           430                   - - Arg Glu Leu Ala Trp Ala Tyr His Ala Val Ty - #r Cys Ser Trp Ile Met               435          - #       440          - #       445                       - - Leu Gln Tyr Ile Thr Val Glu Gln Met Val Gl - #n Leu Cys Val Gln Thr           450              - #   455              - #   460                           - - Thr Asn Ile Pro Glu Ile Cys Phe Arg Ser Va - #l Arg Leu Ala His Lys       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Glu Glu Thr Leu Lys Asn Leu His Glu Gln Se - #r Met Leu Pro Met         Ile                                                                                              485  - #               490  - #               495              - - Thr Gly Val Leu Asp Pro Val Arg His His Pr - #o Val Val Ile Glu Leu                   500      - #           505      - #           510                   - - Cys Phe Cys Phe Phe Thr Glu Leu Arg Lys Le - #u Gln Phe Ile Val Ala               515          - #       520          - #       525                       - - Asp Ala Asp Lys Phe His Asp Asp Val Cys Gl - #y Leu Trp Thr Glu Ile           530              - #   535              - #   540                           - - Tyr Arg Gln Ile Leu Ser Asn Pro Ala Ile Ly - #s Pro Arg Ala Ile Asn       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Trp Pro Ala Leu Glu Ser Gln Ser Lys Ala Va - #l Asn His Leu Glu         Glu                                                                                              565  - #               570  - #               575              - - Thr Cys Arg Val                                                                       580                                                                 - -  - - <210> SEQ ID NO 16                                                   <211> LENGTH: 2193                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 16                                                         - - atgcagggtc tagccttctt ggcggccctt gcatgctggc gatgcatatc gt -              #tgacatgt     60                                                                  - - ggagccactg gcgcgttgcc gacaacggcg acgacaataa cccgctccgc ca -             #cgcagctc    120                                                                  - - atcaatggga gaaccaacct ctccatagaa ctggaattca acggcactag tt -             #tttttcta    180                                                                  - - aattggcaaa atctgttgaa tgtgatcacg gagccggccc tgacagagtt gt -             #ggacctcc    240                                                                  - - gccgaagtcg ccgaggacct cagggtaact ctgaaaaaga ggcaaagtct tt -             #ttttcccc    300                                                                  - - aacaagacag ttgtgatctc tggagacggc catcgctata cgtgcgaggt gc -             #cgacgtcg    360                                                                  - - tcgcaaactt ataacatcac caagggcttt aactatagcg ctctgcccgg gc -             #accttggc    420                                                                  - - ggatttggga tcaacgcgcg tctggtactg ggtgatatct tcgcatcaaa at -             #ggtcgcta    480                                                                  - - ttcgcgaggg acaccccaga gtatcgggtg ttttacccaa tgaatgtcat gg -             #ccgtcaag    540                                                                  - - ttttccatat ccattggcaa caacgagtcc ggcgtagcgc tctatggagt gg -             #tgtcggaa    600                                                                  - - gatttcgtgg tcgtcacgct ccacaacagg tccaaagagg ctaacgagac gg -             #cgtcccat    660                                                                  - - cttctgttcg gtctcccgga ttcactgcca tctctgaagg gccatgccac ct -             #atgatgaa    720                                                                  - - ctcacgttcg cccgaaacgc aaaatatgcg ctagtggcga tcctgcctaa ag -             #attcttac    780                                                                  - - cagacactcc ttacagagaa ttacactcgc atatttctga acatgacgga gt -             #cgacgccc    840                                                                  - - ctcgagttca cgcggacgat ccagaccagg atcgtatcaa tcgaggccag gc -             #gcgcctgc    900                                                                  - - gcagctcaag aggcggcgcc ggacatattc ttggtgttgt ttcagatgtt gg -             #tggcacac    960                                                                  - - tttcttgttg cgcggggcat tgccgagcac cgatttgtgg aggtggactg cg -             #tgtgtcgg   1020                                                                  - - cagtatgcgg aactgtattt tctccgccgc atctcgcgtc tgtgcatgcc ca -             #cgttcacc   1080                                                                  - - actgtcgggt ataaccacac cacccttggc gctgtggccg ccacacaaat ag -             #ctcgcgtg   1140                                                                  - - tccgccacga agttggccag tttgccccgc tcttcccagg aaacagtgct gg -             #ccatggtc   1200                                                                  - - cagcttggcg cccgtgatgg cgccgtccct tcctccattc tggagggcat tg -             #ctatggtc   1260                                                                  - - gtcgaacata tgtataccgc ctacacttat gtgtacacac tcggcgatac tg -             #aaagaaaa   1320                                                                  - - ttaatgttgg acatacacac ggtcctcacc gacagctgcc cgcccaaaga ct -             #ccggagta   1380                                                                  - - tcagaaaagc tactgagaac atatttgatg ttcacatcaa tgtgtaccaa ca -             #tagagctg   1440                                                                  - - ggcgaaatga tcgcccgctt ttccaaaccg gacagcctta acatctatag gg -             #cattctcc   1500                                                                  - - ccctgctttc taggactaag gtacgatttg catccagcca agttgcgcgc cg -             #aggcgccg   1560                                                                  - - cagtcgtccg ctctgacgcg gactgccgtt gccagaggaa catcgggatt cg -             #cagaattg   1620                                                                  - - ctccacgcgc tgcacctcga tagcttaaat ttaattccgg cgattaactg tt -             #caaagatt   1680                                                                  - - acagccgaca agataatagc tacggtaccc ttgcctcacg tcacgtatat ca -             #tcagttcc   1740                                                                  - - gaagcactct cgaacgctgt tgtctacgag gtgtcggaga tcttcctcaa ga -             #gtgccatg   1800                                                                  - - tttatatctg ctatcaaacc cgattgctcc ggctttaact tttctcagat tg -             #ataggcac   1860                                                                  - - attcccatag tctacaacat cagcacacca agaagaggtt gccccctttg tg -             #actctgta   1920                                                                  - - atcatgagct acgatgagag cgatggcctg cagtctctca tgtatgtcac ta -             #atgaaagg   1980                                                                  - - gtgcagacca acctcttttt agataagtca cctttctttg ataataacaa cc -             #tacacatt   2040                                                                  - - cattatttgt ggctgaggga caacgggacc gtagtggaga taaggggcat gt -             #atagaaga   2100                                                                  - - cgcgcagcca gtgctttgtt tctaattctc tcttttattg ggttctcggg gg -             #ttatctac   2160                                                                  - - tttctttaca gactgttttc catcctttat tag       - #                  -       #       2193                                                                      - -  - - <210> SEQ ID NO 17                                                   <211> LENGTH: 730                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 17                                                         - - Met Gln Gly Leu Ala Phe Leu Ala Ala Leu Al - #a Cys Trp Arg Cys         Ile                                                                                1               5 - #                 10 - #                 15              - - Ser Leu Thr Cys Gly Ala Thr Gly Ala Leu Pr - #o Thr Thr Ala Thr Thr                    20     - #             25     - #             30                   - - Ile Thr Arg Ser Ala Thr Gln Leu Ile Asn Gl - #y Arg Thr Asn Leu Ser                35         - #         40         - #         45                       - - Ile Glu Leu Glu Phe Asn Gly Thr Ser Phe Ph - #e Leu Asn Trp Gln Asn            50             - #     55             - #     60                           - - Leu Leu Asn Val Ile Thr Glu Pro Ala Leu Th - #r Glu Leu Trp Thr Ser        65                 - # 70                 - # 75                 - # 80        - - Ala Glu Val Ala Glu Asp Leu Arg Val Thr Le - #u Lys Lys Arg Gln Ser                        85 - #                 90 - #                 95               - - Leu Phe Phe Pro Asn Lys Thr Val Val Ile Se - #r Gly Asp Gly His Arg                   100      - #           105      - #           110                   - - Tyr Thr Cys Glu Val Pro Thr Ser Ser Gln Th - #r Tyr Asn Ile Thr Lys               115          - #       120          - #       125                       - - Gly Phe Asn Tyr Ser Ala Leu Pro Gly His Le - #u Gly Gly Phe Gly Ile           130              - #   135              - #   140                           - - Asn Ala Arg Leu Val Leu Gly Asp Ile Phe Al - #a Ser Lys Trp Ser Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Phe Ala Arg Asp Thr Pro Glu Tyr Arg Val Ph - #e Tyr Pro Met Asn         Val                                                                                              165  - #               170  - #               175              - - Met Ala Val Lys Phe Ser Ile Ser Ile Gly As - #n Asn Glu Ser Gly Val                   180      - #           185      - #           190                   - - Ala Leu Tyr Gly Val Val Ser Glu Asp Phe Va - #l Val Val Thr Leu His               195          - #       200          - #       205                       - - Asn Arg Ser Lys Glu Ala Asn Glu Thr Ala Se - #r His Leu Leu Phe Gly           210              - #   215              - #   220                           - - Leu Pro Asp Ser Leu Pro Ser Leu Lys Gly Hi - #s Ala Thr Tyr Asp Glu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Thr Phe Ala Arg Asn Ala Lys Tyr Ala Le - #u Val Ala Ile Leu         Pro                                                                                              245  - #               250  - #               255              - - Lys Asp Ser Tyr Gln Thr Leu Leu Thr Glu As - #n Tyr Thr Arg Ile Phe                   260      - #           265      - #           270                   - - Leu Asn Met Thr Glu Ser Thr Pro Leu Glu Ph - #e Thr Arg Thr Ile Gln               275          - #       280          - #       285                       - - Thr Arg Ile Val Ser Ile Glu Ala Arg Arg Al - #a Cys Ala Ala Gln Glu           290              - #   295              - #   300                           - - Ala Ala Pro Asp Ile Phe Leu Val Leu Phe Gl - #n Met Leu Val Ala His       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Phe Leu Val Ala Arg Gly Ile Ala Glu His Ar - #g Phe Val Glu Val         Asp                                                                                              325  - #               330  - #               335              - - Cys Val Cys Arg Gln Tyr Ala Glu Leu Tyr Ph - #e Leu Arg Arg Ile Ser                   340      - #           345      - #           350                   - - Arg Leu Cys Met Pro Thr Phe Thr Thr Val Gl - #y Tyr Asn His Thr Thr               355          - #       360          - #       365                       - - Leu Gly Ala Val Ala Ala Thr Gln Ile Ala Ar - #g Val Ser Ala Thr Lys           370              - #   375              - #   380                           - - Leu Ala Ser Leu Pro Arg Ser Ser Gln Glu Th - #r Val Leu Ala Met Val       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Gln Leu Gly Ala Arg Asp Gly Ala Val Pro Se - #r Ser Ile Leu Glu         Gly                                                                                              405  - #               410  - #               415              - - Ile Ala Met Val Val Glu His Met Tyr Thr Al - #a Tyr Thr Tyr Val Tyr                   420      - #           425      - #           430                   - - Thr Leu Gly Asp Thr Glu Arg Lys Leu Met Le - #u Asp Ile His Thr Val               435          - #       440          - #       445                       - - Leu Thr Asp Ser Cys Pro Pro Lys Asp Ser Gl - #y Val Ser Glu Lys Leu           450              - #   455              - #   460                           - - Leu Arg Thr Tyr Leu Met Phe Thr Ser Met Cy - #s Thr Asn Ile Glu Leu       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Gly Glu Met Ile Ala Arg Phe Ser Lys Pro As - #p Ser Leu Asn Ile         Tyr                                                                                              485  - #               490  - #               495              - - Arg Ala Phe Ser Pro Cys Phe Leu Gly Leu Ar - #g Tyr Asp Leu His Pro                   500      - #           505      - #           510                   - - Ala Lys Leu Arg Ala Glu Ala Pro Gln Ser Se - #r Ala Leu Thr Arg Thr               515          - #       520          - #       525                       - - Ala Val Ala Arg Gly Thr Ser Gly Phe Ala Gl - #u Leu Leu His Ala Leu           530              - #   535              - #   540                           - - His Leu Asp Ser Leu Asn Leu Ile Pro Ala Il - #e Asn Cys Ser Lys Ile       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Ala Asp Lys Ile Ile Ala Thr Val Pro Le - #u Pro His Val Thr         Tyr                                                                                              565  - #               570  - #               575              - - Ile Ile Ser Ser Glu Ala Leu Ser Asn Ala Va - #l Val Tyr Glu Val Ser                   580      - #           585      - #           590                   - - Glu Ile Phe Leu Lys Ser Ala Met Phe Ile Se - #r Ala Ile Lys Pro Asp               595          - #       600          - #       605                       - - Cys Ser Gly Phe Asn Phe Ser Gln Ile Asp Ar - #g His Ile Pro Ile Val           610              - #   615              - #   620                           - - Tyr Asn Ile Ser Thr Pro Arg Arg Gly Cys Pr - #o Leu Cys Asp Ser Val       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Ile Met Ser Tyr Asp Glu Ser Asp Gly Leu Gl - #n Ser Leu Met Tyr         Val                                                                                              645  - #               650  - #               655              - - Thr Asn Glu Arg Val Gln Thr Asn Leu Phe Le - #u Asp Lys Ser Pro Phe                   660      - #           665      - #           670                   - - Phe Asp Asn Asn Asn Leu His Ile His Tyr Le - #u Trp Leu Arg Asp Asn               675          - #       680          - #       685                       - - Gly Thr Val Val Glu Ile Arg Gly Met Tyr Ar - #g Arg Arg Ala Ala Ser           690              - #   695              - #   700                           - - Ala Leu Phe Leu Ile Leu Ser Phe Ile Gly Ph - #e Ser Gly Val Ile Tyr       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Phe Leu Tyr Arg Leu Phe Ser Ile Leu Tyr                                                   725  - #               730                                      - -  - - <210> SEQ ID NO 18                                                   <211> LENGTH: 1215                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 18                                                         - - atgttacgag ttccggacgt gaaggctagt ctagtagagg gcgcggcgcg cc -             #tgtcgaca     60                                                                  - - ggcgagcgcg tgtttcacgt cttgacctct ccggcggtgg cggccatggt gg -             #gagtctct    120                                                                  - - aatcctgaag tcccgatgcc actgttgttc gaaaagtttg ggactccgga ct -             #cgtctacc    180                                                                  - - ctgccactct acgcggctag gcacccggaa ctatcgttgc tacggatcat gc -             #tctcaccg    240                                                                  - - cacccctacg cgttaagaag ccacttgtgc gtaggcgaag agaccgcatc tc -             #ttggcgtt    300                                                                  - - tacctgcact ccaagccagt cgtacgcggc cacgaattcg aggacacgca ga -             #tactaccg    360                                                                  - - gagtgccggc tggccataac gagcgaccag tcttatacca actttaagat ta -             #tagatctg    420                                                                  - - ccagcgggat gccgtcgcgt ccccatacac gccgcgaaca agcgtgtcgt ca -             #tcgacgag    480                                                                  - - gccgccaacc gcataaaggt gtttgaccca gagtcgcctt taccgcgtca cc -             #ccataaca    540                                                                  - - ccccgtgccg gtcagaccag atctatactg aaacacaaca tcgcacaggt tt -             #gcgaacgg    600                                                                  - - gatatcgtgt cacttaacac agacaacgag gccgcgtcta tgttctacat ga -             #ttggactc    660                                                                  - - aggcggccga gactcggaga aagcccggtc tgtgacttca acaccgttac ca -             #tcatggag    720                                                                  - - cgtgctaaca actcgataac ttttctaccc aagctaaaac tgaaccggct ac -             #aacacctg    780                                                                  - - ttcctgaagc acgtgttgct gcgcagcatg gggctggaaa acatcgtgtc gt -             #gtttctca    840                                                                  - - tcgctgtacg gcgcagaact tgcccctgcg aaaacacacg agcgggagtt ct -             #tcggcgct    900                                                                  - - ctgctagaaa gactcaaacg tcgggtggag gacgcggtct tctgcctgaa ta -             #ccatagag    960                                                                  - - gatttcccgt ttagggaacc cattcgccaa cccccagatt gttccaaggt gc -             #ttatagaa   1020                                                                  - - gccatggaaa agtactttat gatgtgtagc cccaaagacc gtcaaagcgc cg -             #catggcta   1080                                                                  - - ggtgcagggg tggtcgaact gatatgtgac ggcaatccac tttctgaggt gc -             #tcggattt   1140                                                                  - - cttgccaagt atatgcccat acaaaaagaa tgcacaggaa accttttaaa aa -             #tctacgct   1200                                                                  - - ttattgaccg tctaa              - #                  - #                       - #  1215                                                                   - -  - - <210> SEQ ID NO 19                                                   <211> LENGTH: 404                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 19                                                         - - Met Leu Arg Val Pro Asp Val Lys Ala Ser Le - #u Val Glu Gly Ala Ala         1               5 - #                 10 - #                 15               - - Arg Leu Ser Thr Gly Glu Arg Val Phe His Va - #l Leu Thr Ser Pro Ala                    20     - #             25     - #             30                   - - Val Ala Ala Met Val Gly Val Ser Asn Pro Gl - #u Val Pro Met Pro Leu                35         - #         40         - #         45                       - - Leu Phe Glu Lys Phe Gly Thr Pro Asp Ser Se - #r Thr Leu Pro Leu Tyr            50             - #     55             - #     60                           - - Ala Ala Arg His Pro Glu Leu Ser Leu Leu Ar - #g Ile Met Leu Ser Pro        65                 - # 70                 - # 75                 - # 80        - - His Pro Tyr Ala Leu Arg Ser His Leu Cys Va - #l Gly Glu Glu Thr Ala                        85 - #                 90 - #                 95               - - Ser Leu Gly Val Tyr Leu His Ser Lys Pro Va - #l Val Arg Gly His Glu                   100      - #           105      - #           110                   - - Phe Glu Asp Thr Gln Ile Leu Pro Glu Cys Ar - #g Leu Ala Ile Thr Ser               115          - #       120          - #       125                       - - Asp Gln Ser Tyr Thr Asn Phe Lys Ile Ile As - #p Leu Pro Ala Gly Cys           130              - #   135              - #   140                           - - Arg Arg Val Pro Ile His Ala Ala Asn Lys Ar - #g Val Val Ile Asp Glu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ala Ala Asn Arg Ile Lys Val Phe Asp Pro Gl - #u Ser Pro Leu Pro         Arg                                                                                              165  - #               170  - #               175              - - His Pro Ile Thr Pro Arg Ala Gly Gln Thr Ar - #g Ser Ile Leu Lys His                   180      - #           185      - #           190                   - - Asn Ile Ala Gln Val Cys Glu Arg Asp Ile Va - #l Ser Leu Asn Thr Asp               195          - #       200          - #       205                       - - Asn Glu Ala Ala Ser Met Phe Tyr Met Ile Gl - #y Leu Arg Arg Pro Arg           210              - #   215              - #   220                           - - Leu Gly Glu Ser Pro Val Cys Asp Phe Asn Th - #r Val Thr Ile Met Glu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Arg Ala Asn Asn Ser Ile Thr Phe Leu Pro Ly - #s Leu Lys Leu Asn         Arg                                                                                              245  - #               250  - #               255              - - Leu Gln His Leu Phe Leu Lys His Val Leu Le - #u Arg Ser Met Gly Leu                   260      - #           265      - #           270                   - - Glu Asn Ile Val Ser Cys Phe Ser Ser Leu Ty - #r Gly Ala Glu Leu Ala               275          - #       280          - #       285                       - - Pro Ala Lys Thr His Glu Arg Glu Phe Phe Gl - #y Ala Leu Leu Glu Arg           290              - #   295              - #   300                           - - Leu Lys Arg Arg Val Glu Asp Ala Val Phe Cy - #s Leu Asn Thr Ile Glu       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Asp Phe Pro Phe Arg Glu Pro Ile Arg Gln Pr - #o Pro Asp Cys Ser         Lys                                                                                              325  - #               330  - #               335              - - Val Leu Ile Glu Ala Met Glu Lys Tyr Phe Me - #t Met Cys Ser Pro Lys                   340      - #           345      - #           350                   - - Asp Arg Gln Ser Ala Ala Trp Leu Gly Ala Gl - #y Val Val Glu Leu Ile               355          - #       360          - #       365                       - - Cys Asp Gly Asn Pro Leu Ser Glu Val Leu Gl - #y Phe Leu Ala Lys Tyr           370              - #   375              - #   380                           - - Met Pro Ile Gln Lys Glu Cys Thr Gly Asn Le - #u Leu Lys Ile Tyr Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Leu Leu Thr Val                                                            - -  - - <210> SEQ ID NO 20                                                   <211> LENGTH: 2259                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 20                                                         - - atggcagcgc tcgagggccc cctactactg ccaccgagcg cctccctgac ga -             #cgagtccg     60                                                                  - - cagaccacgt gttatcaagc gacttgggaa tcacagctgg aaatattctg ct -             #gtctggcc    120                                                                  - - accaactcgc acctgcaggc agagctgacc ttagaaggtc ttgataagat ga -             #tgcagccc    180                                                                  - - gagcccacct ttttcgcctg cagagcgata cgcagactac tcctggggga ac -             #gcctccac    240                                                                  - - ccttttatac atcaagaagg gactcttttg ggaaaagtgg gtcgacggta ca -             #gcggcgaa    300                                                                  - - ggtttaataa ttgacggtgg tggagtgttt acgcgcggac agatagacac cg -             #acaactac    360                                                                  - - ctacctgcgg tgggatcatg ggaacttacc gatgattgtg ataaaccctg cg -             #aattcagg    420                                                                  - - gagctacgct cgctgtatct tcccgcgcta ctaacgtgca ccatatgtta ca -             #aagccatg    480                                                                  - - ttcaggatag tgtgcaggta cctggagttc tgggagttcg aacagtgttt tc -             #atgcgttt    540                                                                  - - ctggcggtgt tgccccatag tctacaaccc acaatctatc aaaattattt tg -             #cactcctg    600                                                                  - - gagagcctga agcatctctc gttttcaata atgccacccg catccccaga cg -             #cacagcta    660                                                                  - - cattttttaa agtttaacat cagcagcttc atggccacgt gggggtggca cg -             #gagagctg    720                                                                  - - gtctcgctgc gccgtgccat cgctcacaac gtagagcgac tgcccaccgt gc -             #tgaagaac    780                                                                  - - ctgtcgaaac agagtaagca ccaggacgtc aaggttaacg gacgggatct gg -             #tgggcttt    840                                                                  - - cagctggctc taaaccagct cgtgtcccgt ctgcacgtaa aaatccaacg ca -             #aggacccc    900                                                                  - - ggaccaaagc catacagggt ggtcgtcagt accccagatt gtacctacta tc -             #tagtgtat    960                                                                  - - ccgggcacac cggccatcta cagactcgtc atgtgtatgg cagtggcaga ct -             #gcatcggc   1020                                                                  - - cactcgtgca gcggactgca cccctgcgca aactttttag gcacccacga ga -             #caccgcgt   1080                                                                  - - ctcctggcgg cgacgctttc aagaatccgg tacgcgccga aagaccggcg ag -             #cagccatg   1140                                                                  - - aaaggaaatt tgcaggcgtg cttccaacga tacgcggcca cggacgcgcg ga -             #ctctgggc   1200                                                                  - - agctctacag tgtcagacat gctggaaccc acaaaacacg tcagtttgga aa -             #acttcaag   1260                                                                  - - atcaccatat tcaacaccaa catggtgatt aacactaaga taagctgcca cg -             #ttcctaac   1320                                                                  - - accctgcaaa agactatttt aaacatcccc agattgacca acaattttgt ta -             #tacgaaag   1380                                                                  - - tactccgtaa aggaaccttc ttttaccata agcgtgtttt tttccgacaa ca -             #tgtgtcaa   1440                                                                  - - ggcaccgcaa taaacatcaa catcagtggg gacatgctgc actttctctt cg -             #caatgggt   1500                                                                  - - acgctgaaat gctttctgcc aatcaggcac atatttcctg tatcgatagc aa -             #attggaac   1560                                                                  - - tccacgttgg acctgcacgg actggaaaac cagtacatgg tgagaatggg gc -             #gaaaaaac   1620                                                                  - - gtattttgga ccacaaactt tccatctgtg gtctccagca aggatgggct aa -             #acgtgtcc   1680                                                                  - - tggtttaagg ccgcgacagc cacgatttct aaagtgtacg ggcagcctct tg -             #tggaacag   1740                                                                  - - attcgccacg agctggcgcc cattctcacg gaccagcacg cgcgcatcga cg -             #gaaacaaa   1800                                                                  - - aatagaatat tctccctact tgagcacaga aaccgttccc aaatacagac gc -             #tacacaaa   1860                                                                  - - aggttcctgg agtgtctggt ggaatgctgt tcgtttctca ggcttgacgt gg -             #cttgcatt   1920                                                                  - - aggcgagccg ccgcccgggg cctgtttgac ttctcaaaga agataatcag tc -             #acactaaa   1980                                                                  - - agcaaacacg agtgcgcagt actgggatat aaaaagtgta acctaatccc ga -             #aaatctat   2040                                                                  - - gcccgaaaca agaagaccag gctagacgag ttgggccgca atgcaaactt ca -             #tttcgttc   2100                                                                  - - gtcgccacca cgggtcatcg gttcgccgct ctaaagccac aaattgtccg tc -             #acgccatt   2160                                                                  - - cgcaaactag gcctgcactg gcgccaccga acggccgcgt ccaacgagca ga -             #caccgcca   2220                                                                  - - gccgatcccc gcgtacgttg cgtccgtccg ctggtctaa      - #                       - #  2259                                                                      - -  - - <210> SEQ ID NO 21                                                   <211> LENGTH: 752                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 21                                                         - - Met Ala Ala Leu Glu Gly Pro Leu Leu Leu Pr - #o Pro Ser Ala Ser Leu         1               5 - #                 10 - #                 15               - - Thr Thr Ser Pro Gln Thr Thr Cys Tyr Gln Al - #a Thr Trp Glu Ser Gln                    20     - #             25     - #             30                   - - Leu Glu Ile Phe Cys Cys Leu Ala Thr Asn Se - #r His Leu Gln Ala Glu                35         - #         40         - #         45                       - - Leu Thr Leu Glu Gly Leu Asp Lys Met Met Gl - #n Pro Glu Pro Thr Phe            50             - #     55             - #     60                           - - Phe Ala Cys Arg Ala Ile Arg Arg Leu Leu Le - #u Gly Glu Arg Leu His        65                 - # 70                 - # 75                 - # 80        - - Pro Phe Ile His Gln Glu Gly Thr Leu Leu Gl - #y Lys Val Gly Arg Arg                        85 - #                 90 - #                 95               - - Tyr Ser Gly Glu Gly Leu Ile Ile Asp Gly Gl - #y Gly Val Phe Thr Arg                   100      - #           105      - #           110                   - - Gly Gln Ile Asp Thr Asp Asn Tyr Leu Pro Al - #a Val Gly Ser Trp Glu               115          - #       120          - #       125                       - - Leu Thr Asp Asp Cys Asp Lys Pro Cys Glu Ph - #e Arg Glu Leu Arg Ser           130              - #   135              - #   140                           - - Leu Tyr Leu Pro Ala Leu Leu Thr Cys Thr Il - #e Cys Tyr Lys Ala Met       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Phe Arg Ile Val Cys Arg Tyr Leu Glu Phe Tr - #p Glu Phe Glu Gln         Cys                                                                                              165  - #               170  - #               175              - - Phe His Ala Phe Leu Ala Val Leu Pro His Se - #r Leu Gln Pro Thr Ile                   180      - #           185      - #           190                   - - Tyr Gln Asn Tyr Phe Ala Leu Leu Glu Ser Le - #u Lys His Leu Ser Phe               195          - #       200          - #       205                       - - Ser Ile Met Pro Pro Ala Ser Pro Asp Ala Gl - #n Leu His Phe Leu Lys           210              - #   215              - #   220                           - - Phe Asn Ile Ser Ser Phe Met Ala Thr Trp Gl - #y Trp His Gly Glu Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Ser Leu Arg Arg Ala Ile Ala His Asn Va - #l Glu Arg Leu Pro         Thr                                                                                              245  - #               250  - #               255              - - Val Leu Lys Asn Leu Ser Lys Gln Ser Lys Hi - #s Gln Asp Val Lys Val                   260      - #           265      - #           270                   - - Asn Gly Arg Asp Leu Val Gly Phe Gln Leu Al - #a Leu Asn Gln Leu Val               275          - #       280          - #       285                       - - Ser Arg Leu His Val Lys Ile Gln Arg Lys As - #p Pro Gly Pro Lys Pro           290              - #   295              - #   300                           - - Tyr Arg Val Val Val Ser Thr Pro Asp Cys Th - #r Tyr Tyr Leu Val Tyr       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Pro Gly Thr Pro Ala Ile Tyr Arg Leu Val Me - #t Cys Met Ala Val         Ala                                                                                              325  - #               330  - #               335              - - Asp Cys Ile Gly His Ser Cys Ser Gly Leu Hi - #s Pro Cys Ala Asn Phe                   340      - #           345      - #           350                   - - Leu Gly Thr His Glu Thr Pro Arg Leu Leu Al - #a Ala Thr Leu Ser Arg               355          - #       360          - #       365                       - - Ile Arg Tyr Ala Pro Lys Asp Arg Arg Ala Al - #a Met Lys Gly Asn Leu           370              - #   375              - #   380                           - - Gln Ala Cys Phe Gln Arg Tyr Ala Ala Thr As - #p Ala Arg Thr Leu Gly       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ser Ser Thr Val Ser Asp Met Leu Glu Pro Th - #r Lys His Val Ser         Leu                                                                                              405  - #               410  - #               415              - - Glu Asn Phe Lys Ile Thr Ile Phe Asn Thr As - #n Met Val Ile Asn Thr                   420      - #           425      - #           430                   - - Lys Ile Ser Cys His Val Pro Asn Thr Leu Gl - #n Lys Thr Ile Leu Asn               435          - #       440          - #       445                       - - Ile Pro Arg Leu Thr Asn Asn Phe Val Ile Ar - #g Lys Tyr Ser Val Lys           450              - #   455              - #   460                           - - Glu Pro Ser Phe Thr Ile Ser Val Phe Phe Se - #r Asp Asn Met Cys Gln       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Gly Thr Ala Ile Asn Ile Asn Ile Ser Gly As - #p Met Leu His Phe         Leu                                                                                              485  - #               490  - #               495              - - Phe Ala Met Gly Thr Leu Lys Cys Phe Leu Pr - #o Ile Arg His Ile Phe                   500      - #           505      - #           510                   - - Pro Val Ser Ile Ala Asn Trp Asn Ser Thr Le - #u Asp Leu His Gly Leu               515          - #       520          - #       525                       - - Glu Asn Gln Tyr Met Val Arg Met Gly Arg Ly - #s Asn Val Phe Trp Thr           530              - #   535              - #   540                           - - Thr Asn Phe Pro Ser Val Val Ser Ser Lys As - #p Gly Leu Asn Val Ser       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Trp Phe Lys Ala Ala Thr Ala Thr Ile Ser Ly - #s Val Tyr Gly Gln         Pro                                                                                              565  - #               570  - #               575              - - Leu Val Glu Gln Ile Arg His Glu Leu Ala Pr - #o Ile Leu Thr Asp Gln                   580      - #           585      - #           590                   - - His Ala Arg Ile Asp Gly Asn Lys Asn Arg Il - #e Phe Ser Leu Leu Glu               595          - #       600          - #       605                       - - His Arg Asn Arg Ser Gln Ile Gln Thr Leu Hi - #s Lys Arg Phe Leu Glu           610              - #   615              - #   620                           - - Cys Leu Val Glu Cys Cys Ser Phe Leu Arg Le - #u Asp Val Ala Cys Ile       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Arg Arg Ala Ala Ala Arg Gly Leu Phe Asp Ph - #e Ser Lys Lys Ile         Ile                                                                                              645  - #               650  - #               655              - - Ser His Thr Lys Ser Lys His Glu Cys Ala Va - #l Leu Gly Tyr Lys Lys                   660      - #           665      - #           670                   - - Cys Asn Leu Ile Pro Lys Ile Tyr Ala Arg As - #n Lys Lys Thr Arg Leu               675          - #       680          - #       685                       - - Asp Glu Leu Gly Arg Asn Ala Asn Phe Ile Se - #r Phe Val Ala Thr Thr           690              - #   695              - #   700                           - - Gly His Arg Phe Ala Ala Leu Lys Pro Gln Il - #e Val Arg His Ala Ile       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Arg Lys Leu Gly Leu His Trp Arg His Arg Th - #r Ala Ala Ser Asn         Glu                                                                                              725  - #               730  - #               735              - - Gln Thr Pro Pro Ala Asp Pro Arg Val Arg Cy - #s Val Arg Pro Leu Val                   740      - #           745      - #           750                   - -  - - <210> SEQ ID NO 22                                                   <211> LENGTH: 364                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 22                                                         - - atggtacgtc caaccgaggc cgaggttaag aaatccctga gcaggcttcc ag -              #cagcacgc     60                                                                  - - aaaagagcag gtaaccgggc ccacctggcc acctaccgcc ggctcctcaa gt -             #actccacc    120                                                                  - - ctgcccgatc tatggcggtt tctaagtagc cggccccaga accctcccct tg -             #gacaccac    180                                                                  - - agattattct ttgaggtgac tctagggcac agaattgccg actgcgtaat tc -             #tggtatcg    240                                                                  - - ggtgggcatc agcccgtatg ttacgttgta gagctcaaga cttgtctgag tc -             #accagctg    300                                                                  - - atcccaacca acaccgtgag aacgtcacag cgagctcaag gcctgtgcca ac -             #tctccgac    360                                                                  - - tcga                 - #                  - #                  - #                 364                                                                   - -  - - <210> SEQ ID NO 23                                                   <211> LENGTH: 121                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 23                                                         - - Met Val Arg Pro Thr Glu Ala Glu Val Lys Ly - #s Ser Leu Ser Arg Leu         1               5 - #                 10 - #                 15               - - Pro Ala Ala Arg Lys Arg Ala Gly Asn Arg Al - #a His Leu Ala Thr Tyr                    20     - #             25     - #             30                   - - Arg Arg Leu Leu Lys Tyr Ser Thr Leu Pro As - #p Leu Trp Arg Phe Leu                35         - #         40         - #         45                       - - Ser Ser Arg Pro Gln Asn Pro Pro Leu Gly Hi - #s His Arg Leu Phe Phe            50             - #     55             - #     60                           - - Glu Val Thr Leu Gly His Arg Ile Ala Asp Cy - #s Val Ile Leu Val Ser        65                 - # 70                 - # 75                 - # 80        - - Gly Gly His Gln Pro Val Cys Tyr Val Val Gl - #u Leu Lys Thr Cys Leu                        85 - #                 90 - #                 95               - - Ser His Gln Leu Ile Pro Thr Asn Thr Val Ar - #g Thr Ser Gln Arg Ala                   100      - #           105      - #           110                   - - Gln Gly Leu Cys Gln Leu Ser Asp Ser                                               115          - #       120                                              - -  - - <210> SEQ ID NO 24                                                   <211> LENGTH: 918                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 24                                                         - - atggcactcg acaagagtat agtggttaac ttcacctcca gactcttcgc tg -              #atgaactg     60                                                                  - - gccgcccttc agtcaaaaat agggagcgta ctgccgctcg gagattgcca cc -             #gtttacaa    120                                                                  - - aatatacagg cattgggcct ggggtgcgta tgctcacgtg agacatctcc gg -             #actacatc    180                                                                  - - caaattatgc agtatctatc caagtgcaca ctcgctgtcc tggaggaggt tc -             #gcccggac    240                                                                  - - agcctgcgcc taacgcggat ggatccctct gacaaccttc agataaaaaa cg -             #tatatgcc    300                                                                  - - cccttttttc agtgggacag caacacccag ctagcagtgc tacccccatt tt -             #ttagccga    360                                                                  - - aaggattcca ccattgtgct cgaatccaac ggatttgacc ccgtgttccc ca -             #tggtcgtg    420                                                                  - - ccgcagcaac tggggcacgc tattctgcag cagctgttgg tgtaccacat ct -             #actccaaa    480                                                                  - - atatcggccg gggccccgga tgatgtaaat atggcggaac ttgatctata ta -             #ccaccaat    540                                                                  - - gtgtcattta tggggcgcac atatcgtctg gacgtagaca acacggatcc ac -             #gtactgcc    600                                                                  - - ctgcgagtgc ttgacgatct gtccatgtac ctttgtatcc tatcagcctt gg -             #ttcccagg    660                                                                  - - gggtgtctcc gtctgctcac ggcgctcgtg cggcacgaca ggcatcctct ga -             #cagaggtg    720                                                                  - - tttgaggggg tggtgccaga tgaggtgacc aggatagatc tcgaccagtt ga -             #gcgtccca    780                                                                  - - gatgacatca ccaggatgcg cgtcatgttc tcctatcttc agagtctcag tt -             #ctatattt    840                                                                  - - aatcttggcc ccagactgca cgtgtatgcc tactcggcag agactttggc gg -             #cctcctgt    900                                                                  - - tggtattccc cacgctaa             - #                  - #                       - # 918                                                                   - -  - - <210> SEQ ID NO 25                                                   <211> LENGTH: 305                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 25                                                         - - Met Ala Leu Asp Lys Ser Ile Val Val Asn Ph - #e Thr Ser Arg Leu Phe         1               5 - #                 10 - #                 15               - - Ala Asp Glu Leu Ala Ala Leu Gln Ser Lys Il - #e Gly Ser Val Leu Pro                    20     - #             25     - #             30                   - - Leu Gly Asp Cys His Arg Leu Gln Asn Ile Gl - #n Ala Leu Gly Leu Gly                35         - #         40         - #         45                       - - Cys Val Cys Ser Arg Glu Thr Ser Pro Asp Ty - #r Ile Gln Ile Met Gln            50             - #     55             - #     60                           - - Tyr Leu Ser Lys Cys Thr Leu Ala Val Leu Gl - #u Glu Val Arg Pro Asp        65                 - # 70                 - # 75                 - # 80        - - Ser Leu Arg Leu Thr Arg Met Asp Pro Ser As - #p Asn Leu Gln Ile Lys                        85 - #                 90 - #                 95               - - Asn Val Tyr Ala Pro Phe Phe Gln Trp Asp Se - #r Asn Thr Gln Leu Ala                   100      - #           105      - #           110                   - - Val Leu Pro Pro Phe Phe Ser Arg Lys Asp Se - #r Thr Ile Val Leu Glu               115          - #       120          - #       125                       - - Ser Asn Gly Phe Asp Pro Val Phe Pro Met Va - #l Val Pro Gln Gln Leu           130              - #   135              - #   140                           - - Gly His Ala Ile Leu Gln Gln Leu Leu Val Ty - #r His Ile Tyr Ser Lys       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ile Ser Ala Gly Ala Pro Asp Asp Val Asn Me - #t Ala Glu Leu Asp         Leu                                                                                              165  - #               170  - #               175              - - Tyr Thr Thr Asn Val Ser Phe Met Gly Arg Th - #r Tyr Arg Leu Asp Val                   180      - #           185      - #           190                   - - Asp Asn Thr Asp Pro Arg Thr Ala Leu Arg Va - #l Leu Asp Asp Leu Ser               195          - #       200          - #       205                       - - Met Tyr Leu Cys Ile Leu Ser Ala Leu Val Pr - #o Arg Gly Cys Leu Arg           210              - #   215              - #   220                           - - Leu Leu Thr Ala Leu Val Arg His Asp Arg Hi - #s Pro Leu Thr Glu Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Phe Glu Gly Val Val Pro Asp Glu Val Thr Ar - #g Ile Asp Leu Asp         Gln                                                                                              245  - #               250  - #               255              - - Leu Ser Val Pro Asp Asp Ile Thr Arg Met Ar - #g Val Met Phe Ser Tyr                   260      - #           265      - #           270                   - - Leu Gln Ser Leu Ser Ser Ile Phe Asn Leu Gl - #y Pro Arg Leu His Val               275          - #       280          - #       285                       - - Tyr Ala Tyr Ser Ala Glu Thr Leu Ala Ala Se - #r Cys Trp Tyr Ser Pro           290              - #   295              - #   300                           - - Arg                                                                       305                                                                             - -  - - <210> SEQ ID NO 26                                                   <211> LENGTH: 873                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 26                                                         - - atggcgtcat ctgatattct gtcggttgca aggacggatg acggctccgt ct -              #gtgaagtc     60                                                                  - - tccctgcgtg gaggtaggaa aaaaactacc gtctacctgc cggacactga ac -             #cctgggtg    120                                                                  - - gtagagaccg acgccatcaa agacgccttc ctcagcgacg ggatcgtgga ta -             #tggctcga    180                                                                  - - aagcttcatc gtggtgccct gccctcaaat tctcacaacg gcttgaggat gg -             #tgcttttt    240                                                                  - - tgttattgtt acttgcaaaa ttgtgtgtac ctagccctgt ttctgtgccc cc -             #ttaatcct    300                                                                  - - tacttggtaa ctccctcaag cattgagttt gccgagcccg ttgtggcacc tg -             #aggtgctc    360                                                                  - - ttcccacacc cggctgagat gtctcgcggt tgcgatgacg cgattttctg ta -             #aactgccc    420                                                                  - - tataccgtgc ctataatcaa caccacgttt ggacgcattt acccgaactc ta -             #cacgcgag    480                                                                  - - ccggacggca ggcctacgga ttactccatg gcccttagaa gggcttttgc ag -             #ttatggtt    540                                                                  - - aacacgtcat gtgcaggagt gacattgtgc cgcggagaaa ctcagaccgc at -             #cccgtaac    600                                                                  - - cacactgagt gggaaaatct gctggctatg ttttctgtga ttatctatgc ct -             #tagatcac    660                                                                  - - aactgtcacc cggaagcact gtctatcgcg agcggcatct ttgacgagcg tg -             #actatgga    720                                                                  - - ttattcatct ctcagccccg gagcgtgccc tcgcctaccc cttgcgacgt gt -             #cgtgggaa    780                                                                  - - gatatctaca acgggactta cctagctcgg cctggaaact gtgacccctg gc -             #ccaatcta    840                                                                  - - tccacccctc ccttgattct aaattttaaa taa       - #                  -       #        873                                                                      - -  - - <210> SEQ ID NO 27                                                   <211> LENGTH: 290                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 27                                                         - - Met Ala Ser Ser Asp Ile Leu Ser Val Ala Ar - #g Thr Asp Asp Gly         Ser                                                                                1               5 - #                 10 - #                 15              - - Val Cys Glu Val Ser Leu Arg Gly Gly Arg Ly - #s Lys Thr Thr Val Tyr                    20     - #             25     - #             30                   - - Leu Pro Asp Thr Glu Pro Trp Val Val Glu Th - #r Asp Ala Ile Lys Asp                35         - #         40         - #         45                       - - Ala Phe Leu Ser Asp Gly Ile Val Asp Met Al - #a Arg Lys Leu His Arg            50             - #     55             - #     60                           - - Gly Ala Leu Pro Ser Asn Ser His Asn Gly Le - #u Arg Met Val Leu Phe        65                 - # 70                 - # 75                 - # 80        - - Cys Tyr Cys Tyr Leu Gln Asn Cys Val Tyr Le - #u Ala Leu Phe Leu Cys                        85 - #                 90 - #                 95               - - Pro Leu Asn Pro Tyr Leu Val Thr Pro Ser Se - #r Ile Glu Phe Ala Glu                   100      - #           105      - #           110                   - - Pro Val Val Ala Pro Glu Val Leu Phe Pro Hi - #s Pro Ala Glu Met Ser               115          - #       120          - #       125                       - - Arg Gly Cys Asp Asp Ala Ile Phe Cys Lys Le - #u Pro Tyr Thr Val Pro           130              - #   135              - #   140                           - - Ile Ile Asn Thr Thr Phe Gly Arg Ile Tyr Pr - #o Asn Ser Thr Arg Glu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Pro Asp Gly Arg Pro Thr Asp Tyr Ser Met Al - #a Leu Arg Arg Ala         Phe                                                                                              165  - #               170  - #               175              - - Ala Val Met Val Asn Thr Ser Cys Ala Gly Va - #l Thr Leu Cys Arg Gly                   180      - #           185      - #           190                   - - Glu Thr Gln Thr Ala Ser Arg Asn His Thr Gl - #u Trp Glu Asn Leu Leu               195          - #       200          - #       205                       - - Ala Met Phe Ser Val Ile Ile Tyr Ala Leu As - #p His Asn Cys His Pro           210              - #   215              - #   220                           - - Glu Ala Leu Ser Ile Ala Ser Gly Ile Phe As - #p Glu Arg Asp Tyr Gly       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Phe Ile Ser Gln Pro Arg Ser Val Pro Se - #r Pro Thr Pro Cys         Asp                                                                                              245  - #               250  - #               255              - - Val Ser Trp Glu Asp Ile Tyr Asn Gly Thr Ty - #r Leu Ala Arg Pro Gly                   260      - #           265      - #           270                   - - Asn Cys Asp Pro Trp Pro Asn Leu Ser Thr Pr - #o Pro Leu Ile Leu Asn               275          - #       280          - #       285                       - - Phe Lys                                                                       290                                                                         - -  - - <210> SEQ ID NO 28                                                   <211> LENGTH: 363                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 28                                                         - - atgagcatga ctttccccgt ctccagtcac cggaggaatg gtggacggct cc -              #gtcctggt     60                                                                  - - gcgaatggcc accaagcctc ccgtgattgg tcttataaca gtgctcttcc tc -             #ctagtcat    120                                                                  - - aggcgcctgc gtctactgct gcattcgcgt gttcctggcg gctcgactgt gg -             #cgcgccac    180                                                                  - - cccactaggc agggccaccg tggcgtatca ggtccttcgc accctgggac cg -             #caggccgg    240                                                                  - - gtcacatgca ccgccgacgg tgggcatagc tacccaggag ccctaccgta ca -             #atatacat    300                                                                  - - gccagattag aacggggtgt gtgctataat ggatggctat gggggggggc tg -             #tagataat    360                                                                  - - tga                  - #                  - #                  - #                 363                                                                   - -  - - <210> SEQ ID NO 29                                                   <211> LENGTH: 120                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 29                                                         - - Met Ser Met Thr Phe Pro Val Ser Ser His Ar - #g Arg Asn Gly Gly Arg         1               5 - #                 10 - #                 15               - - Leu Arg Pro Gly Ala Asn Gly His Gln Ala Se - #r Arg Asp Trp Ser Tyr                    20     - #             25     - #             30                   - - Asn Ser Ala Leu Pro Pro Ser His Arg Arg Le - #u Arg Leu Leu Leu His                35         - #         40         - #         45                       - - Ser Arg Val Pro Gly Gly Ser Thr Val Ala Ar - #g His Pro Thr Arg Gln            50             - #     55             - #     60                           - - Gly His Arg Gly Val Ser Gly Pro Ser His Pr - #o Gly Thr Ala Gly Arg        65                 - # 70                 - # 75                 - # 80        - - Val Thr Cys Thr Ala Asp Gly Gly His Ser Ty - #r Pro Gly Ala Leu Pro                        85 - #                 90 - #                 95               - - Tyr Asn Ile His Ala Arg Leu Glu Arg Gly Va - #l Cys Tyr Asn Gly Trp                   100      - #           105      - #           110                   - - Leu Trp Gly Gly Ala Val Asp Asn                                                   115          - #       120                                              - -  - - <210> SEQ ID NO 30                                                   <211> LENGTH: 921                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 30                                                         - - atgctgctca gccgtcacag ggagcgcctt gccgccaacc tggaggagac cg -              #ccaaagac     60                                                                  - - gccggagaga ggtgggaact gagtgccccg acattcacgc gacactgtcc ca -             #aaacggca    120                                                                  - - cggatggcgc acccttttat tggcgtggtg cacagaataa actcatacag tt -             #cggtcctg    180                                                                  - - gaaacatact gcacacggca ccatcccgcc acgcccacgt cagcaaatcc cg -             #acgtggga    240                                                                  - - acccccagac cgtccgagga caacgtcccc gcaaagccgc gcctattgga gt -             #ccctatca    300                                                                  - - acatacttgc agatgcggtg tgtgcgcgag gacgcgcacg tctccacggc cg -             #atcaactg    360                                                                  - - gtcgagtacc aggcgggcag aaaaacacac gactccctgc acgcctgctc tg -             #tctaccgc    420                                                                  - - gaacttcagg cttttctggt taacctttcg tcctttctga acggctgtta cg -             #ttcccggg    480                                                                  - - gtgcactggc tggagccctt ccaacagcag ctagtaatgc acactttttt ct -             #ttttggtt    540                                                                  - - tcaatcaagg ccccacaaaa gacgcaccag ttgtttggat tgtttaagca gt -             #acttcggt    600                                                                  - - ttatttgaaa ctccaaacag tgttttacag acgtttaagc aaaaggcaag cg -             #tattccta    660                                                                  - - ataccaagga gacacggaaa gacatggata gtggtggcga tcatcagcat gc -             #tactggca    720                                                                  - - tccgtagaga acattaacat tgggtacgta gcccaccaaa agcacgtagc ca -             #actccgtg    780                                                                  - - ttcgcggaaa tcataaagac gctttgtcgg tggttccccc ccaaaaattt aa -             #acatcaag    840                                                                  - - aaggagaacg gaaccataat ctacacgcga cccggaggac ggtccagctc gc -             #tgatgtgc    900                                                                  - - gcaacatgct tcaataagaa c           - #                  - #                      921                                                                      - -  - - <210> SEQ ID NO 31                                                   <211> LENGTH: 307                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 31                                                         - - Met Leu Leu Ser Arg His Arg Glu Arg Leu Al - #a Ala Asn Leu Glu Glu         1               5 - #                 10 - #                 15               - - Thr Ala Lys Asp Ala Gly Glu Arg Trp Glu Le - #u Ser Ala Pro Thr Phe                    20     - #             25     - #             30                   - - Thr Arg His Cys Pro Lys Thr Ala Arg Met Al - #a His Pro Phe Ile Gly                35         - #         40         - #         45                       - - Val Val His Arg Ile Asn Ser Tyr Ser Ser Va - #l Leu Glu Thr Tyr Cys            50             - #     55             - #     60                           - - Thr Arg His His Pro Ala Thr Pro Thr Ser Al - #a Asn Pro Asp Val Gly        65                 - # 70                 - # 75                 - # 80        - - Thr Pro Arg Pro Ser Glu Asp Asn Val Pro Al - #a Lys Pro Arg Leu Leu                        85 - #                 90 - #                 95               - - Glu Ser Leu Ser Thr Tyr Leu Gln Met Arg Cy - #s Val Arg Glu Asp Ala                   100      - #           105      - #           110                   - - His Val Ser Thr Ala Asp Gln Leu Val Glu Ty - #r Gln Ala Gly Arg Lys               115          - #       120          - #       125                       - - Thr His Asp Ser Leu His Ala Cys Ser Val Ty - #r Arg Glu Leu Gln Ala           130              - #   135              - #   140                           - - Phe Leu Val Asn Leu Ser Ser Phe Leu Asn Gl - #y Cys Tyr Val Pro Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Val His Trp Leu Glu Pro Phe Gln Gln Gln Le - #u Val Met His Thr         Phe                                                                                              165  - #               170  - #               175              - - Phe Phe Leu Val Ser Ile Lys Ala Pro Gln Ly - #s Thr His Gln Leu Phe                   180      - #           185      - #           190                   - - Gly Leu Phe Lys Gln Tyr Phe Gly Leu Phe Gl - #u Thr Pro Asn Ser Val               195          - #       200          - #       205                       - - Leu Gln Thr Phe Lys Gln Lys Ala Ser Val Ph - #e Leu Ile Pro Arg Arg           210              - #   215              - #   220                           - - His Gly Lys Thr Trp Ile Val Val Ala Ile Il - #e Ser Met Leu Leu Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ser Val Glu Asn Ile Asn Ile Gly Tyr Val Al - #a His Gln Lys His         Val                                                                                              245  - #               250  - #               255              - - Ala Asn Ser Val Phe Ala Glu Ile Ile Lys Th - #r Leu Cys Arg Trp Phe                   260      - #           265      - #           270                   - - Pro Pro Lys Asn Leu Asn Ile Lys Lys Glu As - #n Gly Thr Ile Ile Tyr               275          - #       280          - #       285                       - - Thr Arg Pro Gly Gly Arg Ser Ser Ser Leu Me - #t Cys Ala Thr Cys Phe           290              - #   295              - #   300                           - - Asn Lys Asn                                                               305                                                                             - -  - - <210> SEQ ID NO 32                                                   <211> LENGTH: 1365                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 32                                                         - - atggatgcgc atgctatcaa cgaaagatac gtaggtcctc gctgccaccg tt -              #tggcccac     60                                                                  - - gtggtgctgc ctaggacctt tctgctgcat cacgccatac ccctggagcc cg -             #agatcatc    120                                                                  - - ttttccacct acacccggtt cagccggtcg ccagggtcat cccgccggtt gg -             #tggtgtgt    180                                                                  - - gggaaacgtg tcctgccagg ggaggaaaac caacttgcgt cttcaccttc tg -             #gtttggcg    240                                                                  - - cttagcctgc ctctgttttc ccacgatggg aactttcatc catttgacat ct -             #cggtactg    300                                                                  - - cgcatttcct gccctggttc taatcttagt cttactgtca gatttctcta tc -             #tatctctg    360                                                                  - - gtggtggcta tgggggcggg acggaataat gcgcggagtc cgaccgttga cg -             #gggtatcg    420                                                                  - - ccgccagagg gcgccgtagc ccaccctttg gaggaactgc agaggctggc gc -             #gtgctacg    480                                                                  - - ccggacccgg cactcacccg tggaccgttg caggtcctga ccggccttct cc -             #gcgcaggg    540                                                                  - - tcagacggag accgcgccac tcaccacatg gcgctcgagg ctccgggaac cg -             #tgcgtgga    600                                                                  - - gaaagcctag acccgcctgt ttcacagaag gggccagcgc gcacacgcca ca -             #ggccaccc    660                                                                  - - cccgtgcgac tgagcttcaa ccccgtcaat gccgatgtac ccgctacctg gc -             #gagacgcc    720                                                                  - - actaacgtgt actcgggtgc tccctactat gtgtgtgttt acgaacgcgg tg -             #gccgtcag    780                                                                  - - gaagacgact ggctgccgat accactgagc ttcccagaag agcccgtgcc cc -             #cgccaccg    840                                                                  - - ggcttagtgt tcatggacga cttgttcatt aacacgaagc agtgcgactt tg -             #tggacacg    900                                                                  - - ctagaggccg cctgtcgcac gcaaggctac acgttgagac agcgcgtgcc tg -             #tcgccatt    960                                                                  - - cctcgcgacg cggaaatcgc agacgcagtt aaatcgcact ttttagaggc gt -             #gcctagtg   1020                                                                  - - ttacgggggc tggcttcgga ggctagtgcc tggataagag ctgccacgtc cc -             #cgcccctt   1080                                                                  - - ggccgccacg cctgctggat ggacgtgtta ggattatggg aaagccgccc cc -             #acactcta   1140                                                                  - - ggtttggagt tacgcggcgt aaactgtggc ggcacggacg gtgactggtt ag -             #agatttta   1200                                                                  - - aaacagcccg atgtgcaaaa gacagtcagc gggagtcttg tggcatgcgt ga -             #tcgtcaca   1260                                                                  - - cccgcattgg aagcctggct tgtgttacct gggggttttg ctattaaagc cc -             #gctatagg   1320                                                                  - - gcgtcgaagg aggatctggt gttcattcga ggccgctatg gctag   - #                     1365                                                                         - -  - - <210> SEQ ID NO 33                                                   <211> LENGTH: 454                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 33                                                         - - Met Asp Ala His Ala Ile Asn Glu Arg Tyr Va - #l Gly Pro Arg Cys His         1               5 - #                 10 - #                 15               - - Arg Leu Ala His Val Val Leu Pro Arg Thr Ph - #e Leu Leu His His Ala                    20     - #             25     - #             30                   - - Ile Pro Leu Glu Pro Glu Ile Ile Phe Ser Th - #r Tyr Thr Arg Phe Ser                35         - #         40         - #         45                       - - Arg Ser Pro Gly Ser Ser Arg Arg Leu Val Va - #l Cys Gly Lys Arg Val            50             - #     55             - #     60                           - - Leu Pro Gly Glu Glu Asn Gln Leu Ala Ser Se - #r Pro Ser Gly Leu Ala        65                 - # 70                 - # 75                 - # 80        - - Leu Ser Leu Pro Leu Phe Ser His Asp Gly As - #n Phe His Pro Phe Asp                        85 - #                 90 - #                 95               - - Ile Ser Val Leu Arg Ile Ser Cys Pro Gly Se - #r Asn Leu Ser Leu Thr                   100      - #           105      - #           110                   - - Val Arg Phe Leu Tyr Leu Ser Leu Val Val Al - #a Met Gly Ala Gly Arg               115          - #       120          - #       125                       - - Asn Asn Ala Arg Ser Pro Thr Val Asp Gly Va - #l Ser Pro Pro Glu Gly           130              - #   135              - #   140                           - - Ala Val Ala His Pro Leu Glu Glu Leu Gln Ar - #g Leu Ala Arg Ala Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Pro Asp Pro Ala Leu Thr Arg Gly Pro Leu Gl - #n Val Leu Thr Gly         Leu                                                                                              165  - #               170  - #               175              - - Leu Arg Ala Gly Ser Asp Gly Asp Arg Ala Th - #r His His Met Ala Leu                   180      - #           185      - #           190                   - - Glu Ala Pro Gly Thr Val Arg Gly Glu Ser Le - #u Asp Pro Pro Val Ser               195          - #       200          - #       205                       - - Gln Lys Gly Pro Ala Arg Thr Arg His Arg Pr - #o Pro Pro Val Arg Leu           210              - #   215              - #   220                           - - Ser Phe Asn Pro Val Asn Ala Asp Val Pro Al - #a Thr Trp Arg Asp Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Thr Asn Val Tyr Ser Gly Ala Pro Tyr Tyr Va - #l Cys Val Tyr Glu         Arg                                                                                              245  - #               250  - #               255              - - Gly Gly Arg Gln Glu Asp Asp Trp Leu Pro Il - #e Pro Leu Ser Phe Pro                   260      - #           265      - #           270                   - - Glu Glu Pro Val Pro Pro Pro Pro Gly Leu Va - #l Phe Met Asp Asp Leu               275          - #       280          - #       285                       - - Phe Ile Asn Thr Lys Gln Cys Asp Phe Val As - #p Thr Leu Glu Ala Ala           290              - #   295              - #   300                           - - Cys Arg Thr Gln Gly Tyr Thr Leu Arg Gln Ar - #g Val Pro Val Ala Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Pro Arg Asp Ala Glu Ile Ala Asp Ala Val Ly - #s Ser His Phe Leu         Glu                                                                                              325  - #               330  - #               335              - - Ala Cys Leu Val Leu Arg Gly Leu Ala Ser Gl - #u Ala Ser Ala Trp Ile                   340      - #           345      - #           350                   - - Arg Ala Ala Thr Ser Pro Pro Leu Gly Arg Hi - #s Ala Cys Trp Met Asp               355          - #       360          - #       365                       - - Val Leu Gly Leu Trp Glu Ser Arg Pro His Th - #r Leu Gly Leu Glu Leu           370              - #   375              - #   380                           - - Arg Gly Val Asn Cys Gly Gly Thr Asp Gly As - #p Trp Leu Glu Ile Leu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Lys Gln Pro Asp Val Gln Lys Thr Val Ser Gl - #y Ser Leu Val Ala         Cys                                                                                              405  - #               410  - #               415              - - Val Ile Val Thr Pro Ala Leu Glu Ala Trp Le - #u Val Leu Pro Gly Gly                   420      - #           425      - #           430                   - - Phe Ala Ile Lys Ala Arg Tyr Arg Ala Ser Ly - #s Glu Asp Leu Val Phe               435          - #       440          - #       445                       - - Ile Arg Gly Arg Tyr Gly                                                       450                                                                         - -  - - <210> SEQ ID NO 34                                                   <211> LENGTH: 984                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 34                                                         - - atgtttgctt tgagctcgct cgtgtccgag ggtgacccgg aggtgaccag ta -              #ggtacgtc     60                                                                  - - aagggcgtac aacttgccct ggaccttagc gagaacacac ctggacaatt ta -             #agttgata    120                                                                  - - gaaactcccc tgaacagctt cctcttggtt tccaacgtga tgcccgaggt cc -             #agccaatc    180                                                                  - - tgcagtggcc ggccggcctt gcggccagac tttagtaatc tccacttgcc ta -             #gactggag    240                                                                  - - aagctccaga gagtcctcgg gcagggtttc ggggcggcgg gtgaggaaat cg -             #cactggac    300                                                                  - - ccgtctcacg tagaaacaca cgaaaagggc caggtgttct acaaccacta tg -             #ctaccgag    360                                                                  - - gagtggacgt gggctttgac tctgaataag gatgcgctcc ttcgggaggc tg -             #tagatggc    420                                                                  - - ctgtgtgacc ccggaacttg gaagggtctt cttcctgacg acccccttcc gt -             #tgctatgg    480                                                                  - - ctgctgttca acggacccgc ctctttttgt cgggccgact gttgcctgta ca -             #agcagcac    540                                                                  - - tgcggttacc cgggcccggt gctacttcca ggtcacatgt acgctcccaa ac -             #gggatctt    600                                                                  - - ttgtcgttcg ttaatcatgc cctgaagtac accaagtttc tatacggaga tt -             #tttccggg    660                                                                  - - acatgggcgg cggcttgccg cccgccattc gctacttctc ggatacaaag gg -             #tagtgagt    720                                                                  - - cagatgaaaa tcatagatgc ttccgacact tacatttccc acacctgcct ct -             #tgtgtcac    780                                                                  - - atatatcagc aaaatagcat aattgcgggt caggggaccc acgtgggtgg aa -             #tcctactg    840                                                                  - - ttgagtggaa aagggaccca gtatataaca ggcaatgttc agacccaaag gt -             #gtccaact    900                                                                  - - acgggcgact atctaatcat cccatcgtat gacataccgg cgatcatcac ca -             #tgatcaag    960                                                                  - - gagaatggac tcaaccaact ctaa          - #                  - #                    984                                                                      - -  - - <210> SEQ ID NO 35                                                   <211> LENGTH: 327                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 35                                                         - - Met Phe Ala Leu Ser Ser Leu Val Ser Glu Gl - #y Asp Pro Glu Val Thr         1               5 - #                 10 - #                 15               - - Ser Arg Tyr Val Lys Gly Val Gln Leu Ala Le - #u Asp Leu Ser Glu Asn                    20     - #             25     - #             30                   - - Thr Pro Gly Gln Phe Lys Leu Ile Glu Thr Pr - #o Leu Asn Ser Phe Leu                35         - #         40         - #         45                       - - Leu Val Ser Asn Val Met Pro Glu Val Gln Pr - #o Ile Cys Ser Gly Arg            50             - #     55             - #     60                           - - Pro Ala Leu Arg Pro Asp Phe Ser Asn Leu Hi - #s Leu Pro Arg Leu Glu        65                 - # 70                 - # 75                 - # 80        - - Lys Leu Gln Arg Val Leu Gly Gln Gly Phe Gl - #y Ala Ala Gly Glu Glu                        85 - #                 90 - #                 95               - - Ile Ala Leu Asp Pro Ser His Val Glu Thr Hi - #s Glu Lys Gly Gln Val                   100      - #           105      - #           110                   - - Phe Tyr Asn His Tyr Ala Thr Glu Glu Trp Th - #r Trp Ala Leu Thr Leu               115          - #       120          - #       125                       - - Asn Lys Asp Ala Leu Leu Arg Glu Ala Val As - #p Gly Leu Cys Asp Pro           130              - #   135              - #   140                           - - Gly Thr Trp Lys Gly Leu Leu Pro Asp Asp Pr - #o Leu Pro Leu Leu Trp       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Phe Asn Gly Pro Ala Ser Phe Cys Ar - #g Ala Asp Cys Cys         Leu                                                                                              165  - #               170  - #               175              - - Tyr Lys Gln His Cys Gly Tyr Pro Gly Pro Va - #l Leu Leu Pro Gly His                   180      - #           185      - #           190                   - - Met Tyr Ala Pro Lys Arg Asp Leu Leu Ser Ph - #e Val Asn His Ala Leu               195          - #       200          - #       205                       - - Lys Tyr Thr Lys Phe Leu Tyr Gly Asp Phe Se - #r Gly Thr Trp Ala Ala           210              - #   215              - #   220                           - - Ala Cys Arg Pro Pro Phe Ala Thr Ser Arg Il - #e Gln Arg Val Val Ser       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gln Met Lys Ile Ile Asp Ala Ser Asp Thr Ty - #r Ile Ser His Thr         Cys                                                                                              245  - #               250  - #               255              - - Leu Leu Cys His Ile Tyr Gln Gln Asn Ser Il - #e Ile Ala Gly Gln Gly                   260      - #           265      - #           270                   - - Thr His Val Gly Gly Ile Leu Leu Leu Ser Gl - #y Lys Gly Thr Gln Tyr               275          - #       280          - #       285                       - - Ile Thr Gly Asn Val Gln Thr Gln Arg Cys Pr - #o Thr Thr Gly Asp Tyr           290              - #   295              - #   300                           - - Leu Ile Ile Pro Ser Tyr Asp Ile Pro Ala Il - #e Ile Thr Met Ile Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Glu Asn Gly Leu Asn Gln Leu                                                               325                                                             - -  - - <210> SEQ ID NO 36                                                   <211> LENGTH: 330                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 36                                                         - - ggatccctct gacaaccttc agataaaaaa cgtatatgcc cccttttttc ag -             #tgggacag     60                                                                  - - caacacccag ctagcagtgc tacccccatt ttttagccga aaggattcca cc -             #attgtgct    120                                                                  - - cgaatccaac ggatttgacc ccgtgttccc catggtcgtg ccgcagcaac tg -             #gggcacgc    180                                                                  - - tattctgcag cagctgttgg tgtaccacat ctactccaaa atatcggccg gg -             #gccccgga    240                                                                  - - tgatgtaaat atggcggaac ttgatctata taccaccaat gtgtcattta tg -             #gggcgcac    300                                                                  - - atatcgtctg gacgtagaca acacggatcc         - #                  - #               330                                                                      - -  - - <210> SEQ ID NO 37                                                   <211> LENGTH: 627                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 37                                                         - - ggatccgctg gcaggtgggc gcgcacctcg tcgggtagct tggagacaaa ca -              #gctccagg     60                                                                  - - ccagtccgcg ccgtagcgcc tgcaggtgcc tcaccaccgg ggccgggtca tg -             #cgatctgt    120                                                                  - - ttagtccgga gaagataggg cccttgggaa gccgctgaac cagctccagg gt -             #ctccaaga    180                                                                  - - tgcgcaccgg ttgtcggagc tgtcgcgata gaggttaggg taggtgtccg gt -             #ccgtccgt    240                                                                  - - gggctcaaac ctgcccagac acaccactgt ctgctggggg atcatccttc tc -             #agggagat    300                                                                  - - gcattctttg gaagtagtgg tagagatgga gcagactgcc agggcgttgc ag -             #gagtggtg    360                                                                  - - gcgatggtgc gcaccgtttt taagaaaccc cccagggtgg ggactcccgc tc -             #cctgcagc    420                                                                  - - atctcggcct gctgtacgtc cttggcgaat atgcgacgaa atcggctgtg cg -             #cacggggt    480                                                                  - - cccagggccg gtccggtggc atacaggccg gtgagggccc cctgggtctg tc -             #cgcctgga    540                                                                  - - aacagggtgc tgtgaaacaa caggttgcaa ggccgcgaat acccctctgc ac -             #gctgctgt    600                                                                  - - ggacgtgggt gtatgctccg tggatcc          - #                  - #                 627                                                                      - -  - - <210> SEQ ID NO 38                                                   <211> LENGTH: 233                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: probe           - - <400> SEQUENCE: 38                                                         - - agccgaaagg attccaccat tgtgctcgaa tccaacggat ttgaccccgt gt -              #tccccatg     60                                                                  - - gtcgtgccgc agcaactggg gcacgctatt ctgcagcagc tgttggtgta cc -             #acatctac    120                                                                  - - tccaaaatat cggccggggc cccggatgat gtaaatatgg cggaacttga tc -             #tatatacc    180                                                                  - - accaatgtgt catttatggg gcgcacatat cgtctggacg tagacaacac gg - #a                233                                                                        - -  - - <210> SEQ ID NO 39                                                   <211> LENGTH: 328                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: probe           - - <400> SEQUENCE: 39                                                         - - gaaattaccc acgagatcgc ttccctgcac accgcacttg gctactcatc ag -              #tcatcgcc     60                                                                  - - ccggcccacg tggccgccat aactacagac atgggagtac attgtcagga cc -             #tctttatg    120                                                                  - - attttcccag gggacgcgta tcaggaccgc cagctgcatg actatatcaa aa -             #tgaaagcg    180                                                                  - - ggcgtgcaaa ccggctcacc gggaaacaga atggatcacg tgggatacac tg -             #ctggggtt    240                                                                  - - cctcgctgcg agaacctgcc cggtttgagt catggtcagc tggcaacctg cg -             #agataatt    300                                                                  - - cccacgccgg tcacatctga cgttgcct         - #                  - #                 328                                                                      - -  - - <210> SEQ ID NO 40                                                   <211> LENGTH: 132                                                              <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: probe           - - <400> SEQUENCE: 40                                                         - - aacacgtcat gtgcaggagt gacattgtgc cgcggagaaa ctcagaccgc at -              #cccgtaac     60                                                                  - - cacactgagt gggaaaatct gctggctatg ttttctgtga ttatctatgc ct -             #tagatcac    120                                                                  - - aactgtcacc cg              - #                  - #                       - #      132                                                                   - -  - - <210> SEQ ID NO 41                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 41                                                         - - agccgaaagg attccaccat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 42                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 42                                                         - - gaaattaccc acgagatcgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 43                                                   <211> LENGTH: 23                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 43                                                         - - aacacgtcat gtgcaggagt gac           - #                  - #                     23                                                                       - -  - - <210> SEQ ID NO 44                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 44                                                         - - acagggctgg ttgcccaggg t           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 45                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 45                                                         - - agttgcaaac cagacctcag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 46                                                   <211> LENGTH: 304                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Kaposi's sarcoma-associated herpesvir - #us                     - - <400> SEQUENCE: 46                                                         - - Met Leu Thr Asp Lys Thr Ile Ile Val Ser Le - #u Thr Ser Arg Leu Phe         1               5 - #                 10 - #                 15               - - Ala Asp Glu Ile Thr Lys Leu Gln Lys Lys Il - #e Gly Ser Ile Leu Pro                    20     - #             25     - #             30                   - - Leu Gln Asp Pro His Lys Leu Gln Ser Leu As - #p Thr Leu Gly Leu Asn                35         - #         40         - #         45                       - - Ala Val Cys Ser Arg Asp Val Phe Pro Asp Ty - #r Val His Met Phe Ser            50             - #     55             - #     60                           - - Tyr Leu Ser Lys Cys Thr Leu Ala Ile Leu Gl - #u Glu Val Asn Pro Asp        65                 - # 70                 - # 75                 - # 80        - - Asn Leu Ile Leu Thr Arg Leu Asp Pro Ser Gl - #u Thr Tyr Gln Ile Lys                        85 - #                 90 - #                 95               - - Asn Val Tyr Glu Pro Met Phe Gln Trp Asp Gl - #y Phe Ser Asn Leu Thr                   100      - #           105      - #           110                   - - Val Ile Pro Pro Val Phe Gly Arg Gln Gln Al - #a Thr Val Thr Leu Glu               115          - #       120          - #       125                       - - Ser Asn Gly Phe Asp Leu Val Phe Pro Ser Va - #l Val Pro Ser Asp Leu           130              - #   135              - #   140                           - - Ala Gln Ala Ile Ile Gly Lys Leu Leu Leu Ty - #r Asn Leu Tyr Ser Arg       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Val Glu Ser Asp Pro Glu Ile Asn Ile Gl - #u Glu Val Asn Met         Tyr                                                                                              165  - #               170  - #               175              - - Thr Thr Asn Val Thr His Met Gly Arg His Ty - #r Val Leu Asp Ile Asn                   180      - #           185      - #           190                   - - His Asn Asn Pro Asn Glu Ala Leu Lys Ser Le - #u Asp Asp Leu Ala Val               195          - #       200          - #       205                       - - Tyr Thr Lys Ile Leu Ser Ala Leu Ile Pro Ar - #g Ala Lys Leu Arg Val           210              - #   215              - #   220                           - - Leu Thr Ile Leu Met Arg His Asp Gln His Gl - #u Leu Leu Asp Val Phe       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Arg Gly Ile Val Pro Arg Glu Val Tyr Glu Il - #e Asp Ala Asn Ala         Leu                                                                                              245  - #               250  - #               255              - - Ser Ile Gly Asp Asp Ile Thr Arg Met Thr Th - #r Phe Ile Thr Tyr Leu                   260      - #           265      - #           270                   - - Gln Ser Leu Ser Ser Ile Phe Asn Leu Gly Al - #a Lys Leu His Leu Ser               275          - #       280          - #       285                       - - Ser Tyr Ala Ser Glu Thr Gln Thr Ala Thr Cy - #s Trp Ile Ser Tyr Cys           290              - #   295              - #   300                           - -  - - <210> SEQ ID NO 47                                                   <211> LENGTH: 301                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Epstein Barr Virus                                              - - <400> SEQUENCE: 47                                                         - - Met Asp Leu Lys Val Val Val Ser Leu Ser Se - #r Arg Leu Tyr Thr Asp         1               5 - #                 10 - #                 15               - - Glu Ile Ala Lys Met Gln Gln Arg Ile Gly Cy - #s Ile Leu Pro Leu Ala                    20     - #             25     - #             30                   - - Ser Thr His Gly Thr Gln Asn Val Gln Gly Le - #u Gly Leu Gly Gln Val                35         - #         40         - #         45                       - - Tyr Ser Leu Glu Thr Val Pro Asp Tyr Val Se - #r Met Tyr Asn Tyr Leu            50             - #     55             - #     60                           - - Ser Asp Cys Thr Leu Ala Val Leu Asp Glu Va - #l Ser Val Asp Ser Leu        65                 - # 70                 - # 75                 - # 80        - - Ile Leu Thr Lys Ile Val Pro Gly Gln Thr Ty - #r Ala Ile Lys Asn Lys                        85 - #                 90 - #                 95               - - Tyr Gln Pro Phe Phe Gln Trp His Gly Thr Gl - #y Ser Lys Ser Val Met                   100      - #           105      - #           110                   - - Pro Pro Val Phe Gly Arg Glu His Ala Thr Va - #l Lys Leu Glu Ser Asn               115          - #       120          - #       125                       - - Asp Val Asp Ile Val Phe Pro Met Val Leu Pr - #o Thr Pro Ile Ala Glu           130              - #   135              - #   140                           - - Glu Val Leu Gln Lys Ile Leu Leu Phe Asn Va - #l Tyr Ser Arg Val Val       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Met Gln Ala Pro Gly Asn Ala Asp Met Leu As - #p Val His Met His         Leu                                                                                              165  - #               170  - #               175              - - Gly Ser Val Ser Tyr Leu Gly His His Tyr Gl - #u Leu Ala Leu Pro Glu                   180      - #           185      - #           190                   - - Val Pro Gly Pro Leu Gly Leu Ala Leu Leu As - #p Asn Leu Ser Leu Tyr               195          - #       200          - #       205                       - - Phe Cys Ile Met Val Thr Leu Leu Pro Arg Al - #a Ser Met Arg Leu Val           210              - #   215              - #   220                           - - Arg Gly Leu Ile Arg His Glu His His Asp Le - #u Leu Asn Leu Phe Gln       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Glu Met Val Pro Asp Glu Ile Ala Arg Ile Ar - #g Leu Asp Asp Leu         Ser                                                                                              245  - #               250  - #               255              - - Val Ala Asp Asp Leu Ser Arg Met Arg Val Me - #t Met Thr Tyr Leu Gln                   260      - #           265      - #           270                   - - Ser Leu Ala Ser Leu Phe Asn Leu Gly Pro Ar - #g Leu Ala Thr Ala Ala               275          - #       280          - #       285                       - - Tyr Ser Gln Glu Thr Leu Thr Ala Thr Cys Tr - #p Leu Arg                       290              - #   295              - #   300                           - -  - - <210> SEQ ID NO 48                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 48                                                         - - tccgtgttgt ctacgtccag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 49                                                   <211> LENGTH: 18                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 49                                                         - - aggcaacgtc agatgtga             - #                  - #                       - #  18                                                                    - -  - - <210> SEQ ID NO 50                                                   <211> LENGTH: 23                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 50                                                         - - cgggtgacag ttgtgatcta agg           - #                  - #                     23                                                                       - -  - - <210> SEQ ID NO 51                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 51                                                         - - agcactcgca gggcagtacg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 52                                                   <211> LENGTH: 22                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 52                                                         - - gactcttcgc tgatgaaact gg           - #                  - #                      22                                                                       - -  - - <210> SEQ ID NO 53                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 53                                                         - - tccgtgttgt ctacgtccag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 54                                                   <211> LENGTH: 19                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 54                                                         - - aggcaacgtc agatgtgac             - #                  - #                       - # 19                                                                    - -  - - <210> SEQ ID NO 55                                                   <211> LENGTH: 25                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 55                                                         - - catgggagta cattgtcagg acctc          - #                  - #                    25                                                                       - -  - - <210> SEQ ID NO 56                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 56                                                         - - ggaattatct cgcaggttgc c           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 57                                                   <211> LENGTH: 23                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 57                                                         - - ggcgacattc atcaacctca ggg           - #                  - #                     23                                                                       - -  - - <210> SEQ ID NO 58                                                   <211> LENGTH: 23                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 58                                                         - - atatcatcct gtgcgttcac gac           - #                  - #                     23                                                                     __________________________________________________________________________ 

What is claimed is:
 1. An isolated nucleic acid which is at least 30 nucleotides in length and has a sequence which uniquely defines a herpesvirus associated with kaposi's sarcoma, which herpesvirus is present in and recoverable from the HBL-6 cell line (ATCC Accession No CRL 11762).
 2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid is cDNA.
 3. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid is genomic DNA.
 4. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid is RNA.
 5. The isolated nucleic acid of claim 1 wherein the isolated nucleic acid is labeled with a detectable marker.
 6. A replicable vector comprising the isolated nucleic acid of claim
 1. 7. A nucleic acid of at least 14 nucleotides which specifically hybridizes with the isolated nucleic acid of claim
 1. 8. A nucleic acid of at least 14 nucleotides which specifically hybridizes with a nucleic acid which is complementary to the isolated nucleic acid of claim
 1. 9. The isolated nucleic acid of claim 5, wherein the marker is a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker.
 10. A host cell containing the vector of claim
 6. 11. The vector of claim 6, wherein the vector is a plasmid.
 12. The vector of claim 6, wherein the vector is a cosmid.
 13. The vector of claim 6, wherein the vector is a λ phage.
 14. The vector of claim 6, wherein she vector is a YAC.
 15. The nucleic acid of claim 7, wherein the nucleic acid is DNA.
 16. The nucleic acid of claim 7, wherein the nucleic acid is labeled with a detectable marker.
 17. The cell of claim 10 which is a eucaryotic cell.
 18. The cell of claim 10 which is a bacterial cell.
 19. The nucleic acid of claim 16, wherein the detectable marker is a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker.
 20. A method of diagnosing Kaposi's sarcoma which comprises: (a) obtaining nucleic acids from a tumor lesion of a subject; (b) contacting the nucleic acids from the tumor lesion with the labeled nucleic acid of claim 16 under hybridizing conditions; and (c) determining the presence of hybridized nucleic acids from the tumor lesion, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma.
 21. A method of diagnosing Kaposi's sarcoma which comprises: (a) obtaining nucleic acids from a suitable bodily fluid of a subject; (b) contacting said nucleic acids from the suitable bodily fluid with the labeled nucleic acid of claim 16 under hybridizing conditions; and (c) determining the presence of hybridized nucleic acids from the suitable bodily fluid, the presence of which is indicative of Kaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma.
 22. The method of claim 20 wherein the nucleic acids from the tumor lesion are amplified before step (b).
 23. An isolated herpesvirus associated with kaposi's sarcoma which is present in and recoverable from the HBL-6 cell line (ATCC Accession No CRL 11762).
 24. A nucleic acid of at least 14 nucleotides which hybridizes under high stringency conditions to at least a portion of a nucleotide sequence as shown in FIG. 3A (SEQ ID NO:1). 